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ARTIFICIAL  PARTHENOGENESIS 
AND  FERTILIZATION 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO.,  ILLINOIS 


AgpntH 
THE  ("AMBRIDGE  UNIVERSITY  PRESS 

LONDON  AND  EDINBURGH 

THE  MARUZEN-KABUSHIKI-KAISHA 

TOKYO,  OSAKA,  KYOTO 

KARL  W.  HIERSEMANN 

LEIPZIG 

THE  BAKER  &  TAYLOR  COMPANY 

NEW  YORK 


Artificial  Parthenogenesis 
AND  Fertilization 


BY 


JACQUES  LOEB 

MEMBEE  OF  THE  BOCKEFELLER  INSTITUTE   FOR   MEDICAI.   RESEARCH 


ORIGINALLY  TRANSLATED  FROM  THE  GERMAN 

BY  W.  O.  REDMAN  KING,  B.A.,  ASSISTANT  LECTURER  IN  ZOOLOGY 

AT  THE  UNIVERSITY  OF  LEEDS.  ENGLAND 

SUPPLEMENTED  AND  REVISED  BY  THE  AUTHOR 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,  ILLINOIS 


Copyright  1913  By 
The  University  of  Chicago 


All  Rights  Reserved 


Published  November  1913 


Composed  and  Printed  By 

The  University  of  Cliicago  Press 

Chicago,  Illinois,  U.S.A. 


TO 

PROFESSOR  SVANTE  ARRHENIUS 


Y\\^'\ 


PREFACE 

In  1909  the  author  pubUshed  a  book  entitled  Die  chemische 
Entwicklungserregung  des  tierischen  Eies  (Springer,  Berhn),  in 
which  he  gave  an  account  of  his  experiments  on  artificial 
parthenogenesis.  The  object  of  these  experiments  was  the 
substitution  of  phj'^sicochemical  agencies  for  the  mysterious 
complex  ''living  spermatozoon."  The  book  has  been  trans- 
lated into  English  by  Mr.  W.  O.  R.  King,  but,  owing  to  the 
new  observations  since  made,  the  author  has  found  it  necessarv 
to  revise  and  enlarge  the  translation. 

The  book  gives  a  survey  of  the  methods  by  which  the 
unfertilized  egg  can  be  caused  to  develop  into  an  (^mbryo 
and  the  conclusions  which  can  be  drawn  concerning  the  mechan- 
ism by  which  the  spermatozoon  produces  this  effect.  The 
theory,  which  the  author  published  in  1905  and  1906,  that  at 
least  two  factors  are  involved  in  this  process,  namely,  one 
which  brings  about  a  change  in  the  surface  of  the  egg  (the 
essential  factor),  and  a  second,  corrective  factor,  seems  to 
explain  all  the  phenomena  observed  in  the  new  territory  and 
has  proved  a  reliable  guide. 

In  developing  the  new  field  of  investigation,  the  writer 
endeavored  to  select  those  variables  and  methods  which  would 
lend  themselves  to  a  quantitative  treatment. 

The  problem  of  fertilization  is  intimately  connected  with 
many  different  problems  of  physiology  and  pathology.  We 
may  mention,  among  others,  the  natural  death  of  the  egg  cell 
and  the  prolongation  of  its  life  by  fertilization;  the  fertiliza- 
tion of  the  egg  by  foreign  blood  and  the  innnunity  of  the  egg 
to  blood  of  its  own  species;  the  relations  between  heterogeneous 
hybridization  and  artificial  parthenogenesis,  between  fertiliza- 
tion and  cytolysis,  and  between  permeability  and  physiological 

vii 


v"i  Preface 


efficiency  of  acids  and  bases.  The  facts  recorded  and  discussed 
m  the  book  therefore  go  beyond  the  special  problem  indicated 
by  the  title. 


The  Rockefeller  Institute 

FOR  Medical  Research 

September  6,  1913 


CONTENTS 

PAGE 

I.   Introductory  Remarks       ------         l 

II.   Some  Remarks  on  the  Morphology  of  Development  -       17 

III.  Fertilization  and  Oxidation        -----      23 

IV.  Hydrolytic   Processes    in    the    Germination    of  Oil- 

containing  Seeds    -------30 

V.   Some    Earlier   Observations   on    Natural   Partheno- 
genesis in  Insects  -------      43 

VI.   On  the  History  of  the  Earlier  Experiments  on  Artificial 

Parthenogenesis     -------47 

VII.  The  First  Experiments  upon  the  Osmotic  Activa- 
tion of  the  Unfertilized  Egg  of  the  Sea-Urchin 
(Arbacia)      --------57 

VTII.   The  Improved  Method  of  Artificial  Parthenogenesis 

in  the  Sea-Urchin  Egg 65 

IX.   The  Effect  of  Artificial  Membrane  Formation  on  the 

Egg -        -        -        -      73 

X.   Further  Examples  of  the  Prolongation  of  the  Life  of 

the  Egg  by  Lack  of  Oxygen  -----      So 

XI.   Further  Experiments  on  the  Action  of  the  Hypertonic 

Solution  after  Membrane  Formation       -        -        -      95 

XII.  The  Effect  of  the  Agencies  of  Artificial  Partheno- 
genesis upon  the  Oxidations.  The  Cytological 
Changes  in»the  Parthenogenetic  Egg    -        -        -     113 

XIII.  The  Relative  Physiological  Efficiency  of  Various  Isos- 

motic  Solutions 127 

XIV.  Chemical    Constitution   and    Relative    Physiological 

Efficiency  of  Acids         -        -        -        -        -        -     13.S 

XV.   The  Activation  of  the  Unfertilized  Egg  by  Bases       -     147 

ix 


Contents 


PAGB 


\ 


XVI.  Analysis  of  the  Original  Method  of  Producing  Arti- 
ficial Parthenogenesis  by  Hypertonic  Solutions 
Alone    ---------     159 

XVII.   Membrane  Formation  and  Cytolysis  _        _        -     173 

XVIII.   The  Fertilizing  Effect  of  Foreign  Blood  and  Foreign 

Cell  Extracts  .---...     191 

XIX.   The  Fertilizing  Effect  of  Sperm  Extract     -        -        -    201 

XX.   The  Mechanism  of  the  Formation  of  the  Fertilization 

Membrane 207 

XXI.  Is  Development  of  the  Sea-Urchin  Egg  Possible  with- 
out Membrane  Formation  or  without  the  Second 
(Corrective)  Factor  ?      -        -        -        -        -        -    219 

XXII.   The  Action  of  the  Spermatozoon  upon  the  Egg.     I. 

Heterogeneous  Hybridization  _        _        .        -     225 

XXIII.  The  Action  of  the  Spermatozoon  upon  the  Egg.     II. 

The  Combination  of  Artificial  Parthenogenesis  and 
Fertilization  with  Sperm  in  the  Same  Egg      -        -    233 

XXIV.  Conditions  for  the  Maturation  of  the  Egg  -        -     243 
XXV.   Artificial  Parthenogenesis  in  the  Eggs  of  the  Starfish  -     249 

XXVI.  Artificial  Parthenogenesis  in  the  Eggs  of  Annelids  -  257 

XXVII.  Experiments  with  the  Eggs  of  Molluscs     -        -  -  267 

XXVIII.  Artificial  Parthenogenesis  in  the  Eggs  of  Frogs  -  -  271 

XXIX.  Artificial  Parthenogenesis  in  Plants   -        -        -  -  277 

XXX.   Preservation  of  the  Life  of  the  Egg  by  the  Act  of 

Fertilization  -        -        -        -        -        -        -281 

XXXI.   Artificial  Parthenogenesis  and  Heredity     -        -        -    291 

XXXII.   Can  an  Embryo  Develop  from  a  Spermatozoon  ?       -    303 


INTRODUCTORY   REMARKS 

1.  The  analysis  of  the  mechanism  by  wliich  tlio  male  sex 
cell,  the  spermatozoon,  causes  the  animal  egg  to  develop  is  the 
subject  of  this  book.  This  analysis  was  rendered  possible 
through  the  fact  that  we  are  now  able  to  imitate  the  action  of 
the  spermatozoon  upon  the  egg  by  physicochemical  mean,-. 
The  problem  of  fertilization  is  intimately  connected  with  an- 
other problem,  namely,  that  of  natural  death  and  immortality 
of  the  cell.  Weismann  enunciated  the  fact  that  the  protozoa 
are  immortal;  and  that  the  same  immortality  exists  for  the 
sex  cells  of  metazoa.^ 

The  writer  showed  in  1902  that  the  problem  of  fertilization 
is  intimately  connected  with  the  problem  of  the  prolongation  of 
the  hfe  of  the  egg  cell.  The  unfertilized  mature  egg  dies  in  a 
comparatively  short  time,  which  may  vary  from  a  few  hours  to  a 
few  weeks  according  to  the  species  or  the  conditions  under  whicli 
the  egg  lives.  The  fertilized  egg,  however,  lives  indefinitely,  inas- 
much as  it  gives  rise,  not  only  to  a  new  individual,  but,  theoreti- 
cally at  least,  to  an  endless  series  of  generations.  The  death  of 
the  unfertilized  egg  is  possibly  the  only  clear  case  of  natural 
death  of  a  cell,  i.e.,  of  death  which  is  not  caused  by  external 
injuries,  and  the  act  of  fertilization  is  thus  far  th(>  only  known 
means  by  which  the  natural  death  of  a  cell  can  be  prevented. 
The  two  problems,  fertilization  and  prolongation  of  lif(\  are 
thus  interwoven,  and  the  experiments  on  the  mechanism  of 
fertilization  become  at  the  same  time  studies  on  the  probh^n 
of  natural  death  and  prolongation  of  the  life  of  the  egg  cell. 

1  Leo  Loeb  pointed  out  ten  years  afjo  tluit  liis  exporimonts  on  the  transplanta- 
tion of  cancer  also  proved  the  immortality  of  the  eanerr  erll.  sine.-  the  sanu-  eancfr 
cell  can  be  transplanted  on  endless  generations  of  animals  and  li\e  inden?nt»'lN . 

1 

fROPEHTY  LIBRARY 

N.  C.  State  College 


2        Artificial  Parthenogenesis  and  Fertilization 

The  eggs  of  a  small  number  of  species  can  develop  "spon- 
taneously," but  in  the  majority  of  animals  no  development  is 
possible  until  a  male  sex  cell,  the  spermatozoon,  enters  the  egg. 

2.  The  spermatozoon  is  a  living  motile  organism,  resembling 
a  certain  class  of  protozoa,  the  flagellates.  All  the  mystery 
which  surrounds  the  term  "life"  formerly  surrounded  also  the 
action  of  the  spermatozoon  upon  the  egg.  The  interest  of  the 
experiments  reported  in  this  book  lies  in  the  fact  that  they 
substitute  the  action  of  well-known  chemical  and  physical 
agencies  for  that  of  the  mysterious  complex  called  "living 
spermatozoon." 

The  spermatozoon  has  two  kinds  of  effects  upon  the  egg: 
in  the  first  place,  it  causes  its  development,  and  secondly  it 
transmits  the  paternal  characters  to  the  developing  embryo. 
Now  it  is  probable  that  the  developmental  and  hereditary 
effects  of  the  spermatozoon  are  connected  with  different 
materials  contained  therein.  For  it  is  possible  to  fertilize  the 
eggs  of  the  sea-urchin  with  the  spermatozoa  of  quite  different 
species  or  genera,  e.g.,  starfish,  brittlestars,  holothurians,- 
crinoids,^  and  even  of  moUusca  (Mytilus^  and  Chlorostoma^) . 
Strangely  enough,  however,  all  these  cases  of  heterogeneous 
fertilization  give  rise  to  the  development  of  a  typical  sea- 
urchin  larva,  viz.,  a  pluteus;  hence  the  spermatozoon  exerts 
here  only  a  developmental,  but  no  hereditary,  effect.  These 
experiments  show  that  we  must  distinguish  between  the  develop- 
mental and  the  hereditary  effect  of  the  spermatozoon,  and 
that  each  of  these  effects  depends  probably  upon  different 
materials  in  the  spermatozoon. 

In  this  treatise  we  shall  consider  only  the  developmental 
effect    of    the    spermatozoon;    or    rather,    we    will    describe 

1  Loeb,  Untersuchunnen  zur  kunsllichen  Parthenogenese,  Leipzig,  1906,  pp.  382- 
483;    P Auger's  Archiv,  XClX,  323.  1903;   CIV,  325,  1904. 

2  Godlewski,  Archiv  f.  E ntwicklungs mechanik,  XX,  579,  1906. 

3  Kupelwieser,  Biolog.  Centralbl.,  XXVI,  744,  1906. 

4  Loeb,  Archiv  f.  Entwicklungsmechanik,  XXVI,  476,  1908. 


Introduction 


experiments  through  which  it  is  possible  to  cause,  by  cheiiiiciil 
means,  the  unfertihzed  eggs  of  various  animals  tu  develop  into 

larvae. 

Such  a  study  could  not  be  undertaken  without  applying 
the  methods  of  experimental  and,  more  especially,  quantitative 
research.  As  long  as  the  spermatozoon  was  the  only  means  of 
calling  forth  the  development  of  the  egg  it  was  impossible  to 
undertake  a  physicochemical  analysis  of  this  process.  The 
work  on  artificial  parthenogenesis,  i.e.,  the  substitution  of  well- 
Imown  physicochemical  forces  for  the  spermatozoon,  made 
such  an  analysis  possible. 

The  older  so-called  theories  of  fertilization  were  merely 
metaphors.  The  egg  was  compared  to  a  clock,  and  it  was  said 
that  the  spermatozoon  set  this  clock  in  motion.  Others  said 
that  the  spermatozoon  communicated  to  the  egg  a  pecu- 
liar mode  of  vibration,  and  still  others  maintained  that  the 
spermatozoon  imparted  a  "stimulus"  to  the  egg.  ''Stimulus" 
is  a  technical  term,  but  scientific  problems  are  not  solved  l)y 
mere  acts  of  nomenclature  or  rhetoric. 

With  the  rise  of  cytology  more  definite  ideas  in  regard  to  the 
mechanism  of  fertiUzation  were  expressed.  O.  Hertwig  definiMJ 
fertihzation  as  the  fusion  of  the  sperm  nucleus  with  the  egg 
nucleus.  While  this  fusion  has  a  bearing  upon  the  transmission 
of  the  paternal  characters  to  the  offspring,  it  does  not  give  us 
any  insight  into  the  nature,  of  the  forces  by  which  the  egg 
is  caused  to  develop.  Hertwig's  definition  is  also  incorrect, 
as  was  clearly  demonstrated  by  Boveri  who  foun.l  that  the 
fusion  of  the  egg  and  sperm  nucleus  had  nothin-  at  all  to  do 
with  the  causation  of  the  development  of  the  egg.  For  he  was 
able  to  show  that  an  egg  deprived  of  its  nucleus  can  actually 
develop  into  an  embryo  if  a  spermatozoon  enters  it.  In  this 
case  no  union  of  two  nuclei  takes  place. 

Boveri  replaced  Hertwig's  definition  by  a  hypothesis  tliat 
was  more  in  accordance  with  the  facts.     According  to  him.  the 


Artificial  Parthenogenesis  and  Fertilization 


unfertilized  egg  is  unable  to  develop  because  it  lacks  the  ''organ" 
for  cell  division.  According  to  Boveri,  this  organ  is  the  centro- 
some,  and  it  is  introduced  into  the  egg  by  the  ''middle  piece" 
of  the  spermatozoon.  During  nuclear  division  there  appear 
in  the  egg  radiating  figures,  the  astrospheres,  whose  physical 
nature  is  at  present  unknown.  Some  eggs,  but  by  no  means 
all,  show  at  the  center  of  a  radiating  system  a  granule,  the 
centrosome,  and  in  this  Boveri  sees  the  "organ  of  cell  division." 
According  to  him  the  unfertilized  egg  lacks  this  granule  which  is 
introduced  at  fertilization  by  the  spermatozoon. 

Boveri's  view  that  the  centrosomes  or  astrospheres  cannot 
be  formed  by  the  unfertilized  egg  was  disproved  by  Morgan, 
who  showed,  as  a  sequel  to  my  experiments  on  the  effects  of 
hypertonic  solutions  upon  the  egg,  that  such  treatment  can 
lead  to  the  formation  of  astrospheres  in  the  unfertilized  egg, 
and  that  these  eggs  can  even  begin  to  segment. 

In  certain  eggs,  e.g.  that  of  Chaetopterus,  the  spermatozoon 
has  to  accomplish  another  task  aside  from  fertilizing  the  egg, 
namely,  it  must  bring  about  the  so-called  maturation  of  the 
egg.  This  consists  in  the  reduction  of  the  nucleus  by  two 
subsequent  nuclear  divisions  and  the  throwing-out  of  two  frag- 
ments of  the  nucleus,  the  so-called  polar  bodies.  For  the 
maturation  division,  centrosomes  and  astrospheres  are  also 
required.  Mead  showed  that  in  the  egg  of  Chaetopterus 
the  two  astrospheres  for  the  maturation  division  are  present  in 
the  egg  before  the  spermatozoon  enters  it,  but  that  this  matura- 
tion division  cannot  take  place  unless  a  spermatozoon  does 
enter.  On  the  other  hand,  the  maturation  division  occurred 
without  a  spermatozoon  entering,  if  some  potassium  was  added 
to  the  sea-water.  Hence  the  effect  of  the  spermatozoon  is  in 
this  case  not  due  to  the  introduction  of  a  centrosome  into 
the  egg. 

3.  Investigations  into  the  effects  of  ions  had  led  the  WTiter 
to  believe  that  in  them  w^e  possess  very  potent  factors  in  life 


Introduction 


phenomena,  and  that  with  their  help  we  should  sueceed  in  con- 
trolling such  phenomena  to  a  much  greater  extent  than  by  any 
other  means.  Organic  chemistry  has  thrown  but  little  light 
upon  the  dynamics  of  living  matter:  and  this  may  be  partly  due 
to  the  fact  that  insufficient  attention  has  been  paid  to  the 
electrolytes.  It  appeared  to  me  that  nothing  would  more 
clearly  demonstrate  the  sovereign  role  that  electrolytes  play  in 
the  phenomena  of  life  than  by  causing,  if  possible,  with  their 
help,  unfertihzed  eggs  to  develop  into  larvae.  The  ions  l)y 
whose  aid  I  confidently  expected  to  achieve  success  were  the 
hydroxylions.  I  had  found  that  the  development  of  fertilized 
sea-urchin  eggs  depended  upon  the  reaction  of  the  solution, 
and  that  a  faintly  alkaline  solution  was  more  favorable  than  a 
slightly  acid  solution.  This  I  attributed  to  an  influence  upon 
oxidations  in  the  eggs.  For  I  had  previously  discovered  that 
without  oxygen  the  fertilized  sea-urchin  egg  can  neither  segment 

nor  develop. 

My  first  experiments  toward  causing  unfertilized  eggs  to 
develop  by  means  of  alkali  consisted  in  exposing  the  eggs  to  sea- 
water  to  which  some  sodium  hydrate  had  been  added,  so  as  to 
increase  the  alkalinity.  These  experiments  were  only  partially 
successful.  In  such  sea-water,  the  eggs  divided  only  once  or 
twice  without  developing  into  larvae.  On  the  other  hand  in 
1899  I  succeeded  in  inducing  unfertilized  sea-urchin  eggs  to 
develop  into  larvae  by  exposing  them  for  two  hours  to  hyper- 
tonic sea-water,  i.e.,  sea-water  to  which  had  been  ad(U>d 
sufficient  salt  or  sugar  to  raise  its  concentration  about  60  pvr 
cent.  Even  pure  (hypertonic)  cane-sugar  solutions  were  found 
to  induce  development,  but  the  larvae  obtained  in  this  ca^e 
were  unable  to  develop  to  the  pluteus  stage.  Six  yc^ars  later  1 
found  that,  within  certain  limits,  the  devel()i)m(Mital  effect  of 
the  hypertonic  solution  increases  with  the  concentration  of  the 
hydroxylions.  Moreover  the  hypertonic  solution  is  only  able 
to  produce  its  developmental  effect  if  it  contains  hcv  oxygen 


6        Artificial  Parthenogenesis  and  Fertilization 


in  sufficient  concentration.  If  the  oxygen  is  driven  out  of  the 
hypertonic  solution,  or  if  the  oxidations  in  the  egg  are  prevented 
by  the  addition  of  some  KCN  to  the  sea-water,  the  develop- 
mental effect  of  the  hypertonic  solution  is  lacking. 

Shortlj^  after  obtaining  larvae  from  unfertilized  sea-urchin 
eggs  by  means  of  hj^pertonic  sea-water,  I  was  able  to  obtain 
swimming  larvae  from  the  unfertilized  eggs  of  Chaetopterus  by 
the  use  of  potassium  and  acids,  and  of  starfish  eggs  by  means 
of  acids,  without  it  being  necessary  to  increase  the  osmotic 
pressure  of  the  sea-water. 

With  these  experiments  one  task  was  accomplished,  namely, 
the  substitution  of  phj^sicochemical  agencies  for  the  mysterious 
complex  ''living  spermatozoon."  But  these  experiments  did 
not  yet  allow  us  to  draw  any  conclusions  concerning  the 
nature  of  the  process  of  fertilization. 

4.  Ever}^  biologist  knew  that  the  eggs  of  many  marine  ani- 
mals underwent  a  characteristic  change  immediately  after 
the  entrance  of  the  spermatozoon,  namely,  the  formation  of  the 
so-called  fertilization  (vitelline)  membrane.  This  phenomenon 
was  always  considered  to  be  something  of  very  secondary 
importance,  and  I  therefore  attached  no  significance  to  the 
fact  that  after  the  osmotic  treatment  the  egg  formed  no  typical 
fertilization  membrane.  However,  in  1905  I  discovered  that  a 
short  exposure  of  the  sea-urchin  egg  to  a  monobasic  fatty  acid,  or 
to  CO2,  led  to  the  formation  of  a  typical  fertilization  membrane 
by  all  the  eggs  of  Strongylocentrotus  purpiiratus;  and,  moreover, 
that  all  these  eggs  could  be  induced  to  develop  into  larvae  after 
artificial  membrane  formation  by  a  subsequent  short  exposure, 
some  thirty  to  fifty  minutes,  to  hypertonic  sea-water.  If  mem- 
brane formation  alone  is  evoked,  without  subsequent  exposure 
of  the  eggs  to  hypertonic  sea-water,  they  begin  to  segment,  but 
then  disintegration  sets  in;  and  the  higher  the  temperature,  the 
sooner  does  this  disintegration  make  its  appearance.  If  the 
temperature  is  verj^  low,  the  eggs  can  develop  to  early  larval 


Introduction 


stages  without  it  being  necessary  to  expose  them  in  addition  to 
hypertonic  sea-water.  At  room  temperature,  on  the  other 
hand,  the  eggs  after  artificial  membrane  formation  go  to  pieces 
in  the  course  of  a  few  hours.  Hence  it  is  the  artificial  membrane 
formation  which  starts  the  development,  but  starts  at  the  same 
time  a  tendency  to  disintegration.  The  latter,  however,  can 
be  counteracted  by  a  short  exposure  of  the  eggs  to  a  hypertonic 
solution;  but  in  order  to  produce  this  effect  the  hypertonic 
solution  must  contain  a  sufficient  quantity  of  free  ox3'gen,  and 
the  higher  the  concentration  of  hydroxylions,  within  certain 
limits,  the  more  effective  is  the  hypertonic  solution. 

In  1906  I  discovered  still  another  method  of  overcoming  the 
injurious  secondary  effect  connected  with  membrane  formation, 
which  consists  in  arresting  the  development  of  the  eggs  for  two 
or  three  hours.  This  is  effected  by  putting  the  eggs  after  mem- 
brane formation  into  sea-water  from  which  the  oxygen  has  been 
displaced  by  a  current  of  hydrogen,  or  to  which  some  KCN  has 
been  added.  As  long  as  the  eggs  are  in  such  a  solution  they 
cannot  develop  on  account  of  the  cessation  of  their  oxidations. 
If  the  eggs  are  replaced  some  two  or  three  hours  later  (at  15°  C.) 
in  normal  aerated  sea-water,  practically  all  the  eggs  segment 
and  develop  in  a  perfectly  normal  manner.  Hence  there  must 
have  occurred  during  that  time  a  change  in  the  egg  which  allows 
it  to  develop  normally. 

Further  proof  can  be  given  that  membrane  formation  is 
really  the  essential  step  in  the  activation  of  develo})ment; 
for  in  the  eggs  of  many  forms  membrane  formation  is  sufficient 
to  allow  them  to  develop  into  normal  larvae,  at  room  tempera- 
ture. I  found  that  if  membrane  formation  is  produced  in  the 
eggs  of  a  starfish,  Asterina,  by  the  use  of  a  fatty  acid,  some 
of  the  eggs  are  able  to  develop  into  normal  larvae;  and  I 
afterward  found  the  same  to  be  the  case  with  Polynoc,  a  poly- 
chaet,  while  Lefevre  established  the  same  fact  for  the  eggs  of 
another  marine  worm,  Thalassema.     Now  these  eggs  differ  from 


8        Artificial  Parthenogenesis  and  Fertilization 


those  of  the  sea-urchin  only  in  that  either  the  secondarj-  effects 
set  up  by  membrane  formation  are  less  injurious,  or  that  the 
eggs  of  these  forms  can  recover  more  quickly  from  them  than 
sea-urchin  eggs.  We  shall  see  later  that  the  eggs  of  different 
forms  vary  in  this  respect  quantitatively,  but  not  in  principle. 
It  can  be  shown  in  still  another  way  that  it  is  the  process 
of  membrane  formation  and  not  any  other  effect  of  the  fatty 
acid  which  starts  the  development  of  the  egg.  For  membrane 
formation  induced  in  any  other  way  always  evokes  develop- 
ment, whereas  mere  exposure  to  the  fatty  acid  does  not  lead 
to  development  unless  membrane  formation  also  takes  place. 

5.  In  this  way,  the  process  of  membrane  formation,  which 
had  hitherto  been  regarded  as  of  quite  secondary  importance  so 
far  as  development  was  concerned,  was  identified  as  the  immedi- 
ate cause  of  the  activation  of  development  in  the  egg.  The 
next  step  was  to  determine  the  substances  and  processes  by 
which  membrane  formation  might  be  evoked. 

I  had  already  observed  in  1904  that  whenever  an  egg  under- 
goes cytolysis  it  passes  through  a  similar  process  of  membrane 
formation,  and  further  research  along  this  line  showed  that 
indeed  all  cytolytic  agencies  cause  membrane  formation.  A 
relatively  short  exposure  to  a  cytolytic  agent  leads  only  to 
membrane  formation,  whereas  a  longer  exposure  entails  cytolysis. 
Hence  we  can  say  that  membrane  formation  (and  the  activation 
of  development)  is  due  to  a  cytolysis  of  the  surface  or  the  cortical 
layer  of  the  egg.  Certain  glucosides,  such  as  saponin,  solanin, 
and  digitalin,  produce  strong  haemolytic  or  cytolytic  effects.  If 
eggs  are  exposed  for  a  short  time  to  a  very  dilute  solution  of 
these  substances  in  sea-water,  they  form  a  typical  fertilization 
membrane,  and,  if  removed  from  the  solution  immediately 
after  the  formation  of  this  membrane,  they  will  begin  to  develop. 
For  the  eggs  of  Polynoe  this  treatment  is  sufficient  to  induce 
the  development  to  the  larval  stage;  but  the  eggs  of  Strongylo- 
centrotus  require  a  subsequent  exposure  to  hypertonic  sea-water 


Introduction 


in  order  to  counteract  the  injurious  secondary  effects  of 
membrane  formation.  If  the  eggs  are  not  removed  from  the 
saponin  solution  immediately  after  membrane  formation, 
cytolysis  supervenes  in  a  few  minutes.  Similar  results  are 
obtained  with  soap.  A  short  exposure  of  the  eggs  to  an 
alkaline  soap  solution  in  NaCl  leads  to  membrane  formation 
(and  to  development  if  the  eggs  are  subsequently  treated  for  a 
short  while  with  a  hypertonic  solution).  A  longer  exposure  of 
the  eggs  to  a  soap  solution  leads  to  cytolysis. 

The  same  behavior  can  be  demonstrated  for  all  cytolytic 
reagents,  even  those  of  a  physical  nature,  as,  for  example,  rise 
of  temperature.  A  sufficient  increase  of  temperature  causes 
membrane  formation  in  the  unfertilized  sea-urchin  egg  and  one 
of  longer  duration  leads  to  cytolysis.  R.  Lillie  has  found  that 
the  eggs  of  starfish  can  develop  into  larvae  after  membrane 
formation  caused  by  raising  the  temperature.  Sea-urchin 
eggs  are  too  much  injured  by  the  increase  of  temperature  neces- 
sary for  membrane  formation  to  be  able  to  develop. 

We  know"  that  the  blood  corpuscles  of  any  species  of  animal 
are  often  cytolyzed  by  the  body  fluids  of  different  species.  In 
1907  I  found  that  the  blood  of  certain  worms,  to  wit,  the 
Gephyrea,  causes  membrane  formation  in  the  sea-urchin  egg. 
even  when  greatly  diluted.  This  power  is  also  possessed  by  the 
blood  of  other  forms,  especially  of  mammals.  This  phenomenon 
is,  however,  not  exhibited  by  the  eggs  of  every  female,  and  I 
believe  that  this  is  due  to  a  difference  in  the  permeability  of  the 
eggs  of  different  females.  Only  those  eggs  that  are  permeable 
to  the  ''lysins"  of  the  foreign  blood  form  membranes  under  its 
influence.  If  such  eggs  are  exposed  for  a  short  while  to  hyjier- 
tonic  sea-w^ater,  after  membrane  formation  has  been  produced  by 
the  blood,  they  develop  into  larvae.  The  cytolysis  of  these  eggs 
with  foreign  blood  is  impossible,  or  proceeds  only  very  slowly: 
this,  I  think,  is  due  to  the  fact  that  the  fertilization  membrane 
prevents  the  further  diffusion  of  the  "lysins"  into  the  egg. 


10      Artificial  Parthenogenesis  and  Fertilization 


The  most  remarkable  fact,  however,  is  this:  that  it  is 
possible  to  cause  artificial  parthenogenesis  in  eggs  which  are 
refractory  to  any  other  method  of  artificial  parthenogenesis. 
All  attempts  to  cause  artificial  parthenogenesis  in  the  eggs  of 
Cumingia,  a  marine  mollusc,  had  failed.  But  Mr.  Wasteneys 
and  the  writer  found  in  1912  that  these  eggs  can  be  caused 
to  develop  into  larvae  if  they  are  treated  with  ox  blood  or 
serum.  In  order  to  accompHsh  this  they  must  first  be  sensitized 
by  a  treatment  with  a  solution  of  SrClg.  The  writer  had 
previously  found  that  sea-urchin  eggs  which  cannot  be  caused 
to  form  membranes  under  the  influence  of  ox  serum  will 
do  so  if  they  are  first  treated  for  some  time  with  a  solution 
of  SrCl^. 

Not  only  foreign  blood  but  the  extract  of  foreign  cells  is 
efficient  in  inducing  membrane  formation  and  development  in 
the  unfertiUzed  egg.  Blood  of  the  species  to  which  the  eggs 
belong  is,  however,  entirely  ineffective  for  this  purpose.  This 
is  analogous  to  the  fact  that  foreign  ^'lysins"  may  destroy  the 
cells  of  an  animal  while  the  cells  are  immune  against  the  lysins 
of  their  own  species.  The  experiments  on  artificial  partheno- 
genesis indicate  that  this  immunity  is  due  to  the  fact  that  the 
lysins  are  prevented  from  diffusing  into  the  cells  of  the  same 
species  while  they  can  diffuse  into  the  cells  of  foreign  species. 

6.  The  question  may  next  be  raised  whether  the  spermato- 
zoon also  effects  membrane  formation  by  means  of  a  cytolytic 
substance,  a  "lysin,"  and  whether,  in  that  event,  it  must  carry 
into  the  egg  yet  another  substance  besides  the  lysin  for  the 
purpose  of  preventing  the  disintegration  following  membrane 
formation.  This  is  what  actually  appears  to  be  the  case. 
Ten  years  ago  I  discovered  a  method  of  fertilizing  the  eggs  of  the 
sea-urchin  with  the  sperm  of  widely  different  species,  e.g.,  of 
the  starfish.  In  employing  this  method,  I  found  that  not  all  the 
eggs  of  the  sea-urchin  which  form  membranes  with  (living)  star- 
fish sperm  develop.     Some  of  the  eggs  form  membranes  and 


Introduction  il 


begin  to  develop,  but  then  disintegrate  unless  the  harmful 
secondary  effects  of  membrane  formation  are  counteracted 
by  some  further  manipulation.  Hence  some  of  the  sea-urchin 
eggs  treated  with  starfish  sperm  behave  as  though  membrane 
formation  had  only  been  caused  by  virtue  of  some  haemolytic 
substance  carried  by  the  sperm.  But  if  these  eggs  are  first 
treated  with  starfish  sperm  and  then,  after  membrane  forma- 
tion, exposed  for  a  short  while  to  hypertonic  sea-water,  they 
can  develop  into  larvae.  The  other  sea-urchin  eggs,  however, 
that  formed  membranes  with  starfish  sperm  developed  into 
larvae  without  the  necessity  for  any  further  exposure  to  hyper- 
tonic sea-water  or  to  lack  of  oxygen. 

These  facts  are  intelligible  on  the  following  assumption. 
The  spermatozoon  causes  membrane  formation  by  a  substance 
which  is  comparatively  soluble  in  the  egg;  in  addition  to  this 
membrane-forming  substance  the  spermatozoon  carries  a 
second  substance  into  the  egg  which  prevents  the  disintegration 
which  follows  after  mere  membrane  formation.  Those  eggs 
of  the  sea-urchin  which  upon  contact  with  the  sperm  of  the 
starfish  formed  membranes  but  then  began  to  disintegrate 
absorbed  only  this  membrane-forming  substance;  while  into 
those  eggs  which  formed  a  membrane  and  developed  into  larvae 
the  whole  spermatozoon  had  entered. 

These  observations  upon  the  effect  of  starfish  sperm  on  the 
sea-urchin  egg  lead  to  the  interesting  problem  of  why  the  treat- 
ment of  sea-urchin  eggs  with  their  own  sperm  never  causes 
membrane  formation  alone  (without  complete  development 
following).  For  when  sea-urchin  eggs  are  fertilized  with  sea- 
urchin  sperm,  all  the  eggs  that  form  membranes  invariably 
develop,  and  it  never  happens  that  some  begin  to  develop  and 
afterward  decompose.  The  answer  is  that  the  "lysins"  of  for- 
eign sperm  can  penetrate  into  the  egg  in  two  ways,  by  diffusion 
or  by  being  carried  by  a  spermatozoon;  the  'Mysins"  of  the 
spermatozoa  of  the  same  species,  however,  never  can  get  into 


12       Artificial  Parthenogenesis  and  Fertilization 


the  egg  by  diffusion,  but  only  if  carried  into  the  egg  by  a  Hving 
spermatozoon.  And  when  a  spermatozoon  enters  the  egg  it 
introduces  also  into  the  egg  the  second  substance  which  pre- 
vents the  disintegration  following  membrane  formation. 

7.  The  question  as  to  how  cytolytic  agents  cause  membrane 
formation  is  connected  with  that  of  the  nature  of  cytolysis 
itself.  We  shall  not  in  this  treatise  consider  the  answer  to  this 
question,  but  in  order  to  fix  our  ideas  provisionally,  we  may 
assume  that  the  surface  of  the  egg  consists  of  an  emulsion 
whose  stability  is  destroyed  by  cytolytic  agents.  This  sup- 
position gives  us  an  explanation  of  the  fact  that  the  eggs 
of  some  animals  show  a  slight  natural  tendency  to  parthe- 
nogenesis. In  such  eggs  the  stability  of  the  emulsion  may 
be  relatively  small,  so  that  the  HO  ions  of  the  sea-water, 
or  the  carbonic  acid  produced  by  the  eggs  or  by  bacteria, 
are  in  themselves  sufficient  to  destroy  the  emulsion  and  to 
cause  membrane  formation.  This  hypothesis  is  supported 
by  observations  upon  the  eggs  of  starfish.  Unlike  most  sea- 
urchin  eggs,  those  of  starfish  show  occasionally  a  tendency  to 
develop  spontaneously  (without  the  addition  of  sperm).  As 
a  rule  one  finds  that  a  few  starfish  eggs  begin  to  divide  after 
lying  for  some  time  in  sea-water,  and  in  many  cases  this  is 
followed  by  the  spontaneous  development  to  the  larval  stage 
without  the  necessity  for  any  artificial  manipulation.  Mathews 
found  that  the  number  of  such  spontaneously  developing 
starfish  eggs  can  be  increased  by  slight  mechanical  agitation. 
I  found  the  same  to  be  the  case  for  the  eggs  of  Amphitrite,  a 
marine  worm,  which  also  show  a  tendency  toward  spontaneous 
parthenogenesis.  These  facts,  of  which  there  had  hitherto  been 
no  explanation,  can  be  understood  upon  the  assumption  that 
this  parthenogenetic  tendency  depends  upon  the  slight  stability 
of  the  emulsion  at  the  surface  of  the  eggs  of  these  forms.  At 
the  lower  limit  of  this  stability,  a  shght  shaking  is  enough  to 
destroy  it.     I  have  also  found  that  if  the  eggs  of  Asterias  are 


Introduction  13 


compressed  in  the  ovary,  they  can  be  caused  to  form  membranes 
and  to  cytolyze.  Moreover,  starfish  eggs  that  are  caused  to 
develop  by  shaking  form  membranes  first  of  all;  this  is  the 
immediate  effect  of  the  mechanical  agitation. 

8.  We  may  now  raise  the  question  as  to  how  the  artificial 
membrane  formation  induces  the  development  of  the  egg.     The 
writer  reached  the  conclusion  that  for  the  sea-urchin  egg  this 
was  due  to  an  acceleration  of  oxidations.     This  suggestion  was 
confirmed  by  Warburg,  and  since  by  Wasteneys  and  the  writer. 
The  entrance  of  the  spermatozoon  raises  the  rate  of  oxidations  in 
the  egg  of  the  sea-urchin  to  from  four  to  six  times  its  usual 
amount;   and  the  artificial  membrane  formation  has  the  same 
effect.     There  are  two  possibilities  by  which  this  result  can  be 
produced:    either  a  catalyzer  (an  oxidase)  is  carried  into  the 
egg  by  the  spermatozoon;   or  the  change  in  the  surface  layer 
itself    causes    the    increased    rate    of    oxidation.     Ever>i:hing 
speaks  in  favor  of  the  second  assumption.     We  know  that  with 
the  concentration  of  the  catalyzer  the  rate  of  chemical  reactions 
increases  either  in  proportion  to  the  concentration  of  the  cata- 
lyzer or  in  the  ratio  of  the  square  root  of  the  concentration; 
and  the  investigations  of  the  temperature  coefficient  of  develop- 
ment of  the  egg  show  that  the  rate  of  development  is  deter- 
mined by  chemical  reactions.     If  therefore  the  spermatozoon 
increased  the  rate  of  oxidation  by  carrying  an  oxidase  into  the 
egg  the  rate  of  segmentation  in  an  egg  fertilized  by  more  than 
one  spermatozoon  should  be  increased  in  the  ratio  of  1:2  or 
l:i/2.     Observations  show  that  the  velocity  of  segmentation 
in  eggs  fertiUzed  by  two  spermatozoa  is  identical  with  that 
found  in  eggs  fertilized  by  one  spermatozoon.     This  fact  proves 
that  the  spermatozoon  causes  development,  not  by  carrying  an 
oxidase  or  some  other  catalyzer  into  the  egg,  but  by  removing 
an  obstacle  to  development.     The  same  must  hold  for  the 
explanation   of   the   influence   of   mt^nbrane   formation   upon 
development.     Through    the    cytolysis    of    the    cortical    layer 


14      Artificial  Parthenogenesis  and  Fertilization 

of  the  egg  the  oxidations  in  the  unfertihzed  egg  are  accelerated 
from  four  to  six  times  their  natural  rate. 

This  idea  is  further  supported  by  the  fact  observed  by 
Wasteneys  and  the  writer  that  if  we  cause  complete  cytolysis 
of  the  unfertilized  eggs  of  the  sea-urchin  by  saponin  or  by 
distilled  water,  the  rate  of  oxidation  is  thereby  raised  to  the 
same  amount  as  if  the  eggs  were  fertilized  by  sperm,  although 
these  eggs  are  dead.  This  suggests  the  idea  that  the  cytolysis 
of  the  cortical  layer  removes  an  obstacle  by  which  the  oxidations 
become  possible.  It  may  be  that  the  cortical  layer  of  the 
unfertilized  egg  contains  an  oxidase  which  cannot  act,  or  an 
oxidizable  substance  which  cannot  readily  undergo  oxidation, 
unless  the  cortical  layer  is  liquefied  (cytolyzed).  R.  Lillie 
assumes  that  the  cortical  layer  of  the  unfertilized  egg  prevents 
the  diffusion  of  CO2  from  the  egg  and  that  this  CO2  prevents 
oxidation.  This  view  meets  with  the  difficulty  that  CO2  is  a 
good  agency  for  calling  forth  the  membrane  formation  in  un- 
fertilized eggs,  and  we  shall  see  that  only  such  substances  can 
do  this  as  diffuse  into  the  egg. 

9.  The  earlier  biologists  had  realized  that  the  unfertilized  egg 
dies  quickly  and  that  the  act  of  fertilization  prevents  its  death. 
The  question  arises:  How  can  the  spermatozoon  accomplish 
this  act  ?  The  causation  of  development  requires  two  factors, 
the  one  is  the  membrane-forming  factor,  the  other  the  corrective 
factor  which  in  the  methods  of  artificial  parthenogenesis  is 
supplied  by  the  hypertonic  solution.  We  may  raise  the  question : 
Is  only  one  of  these  factors  responsible  for  the  prolongation 
of  the  life  of  the  egg,  or  are  both  responsible  ?  At  first  sight  it 
would  seem  as  if  the  second  factor  alone  were  responsible  for 
this  effect,  since  the  membrane  formation  alone  raises  the  rate 
of  oxidations  and  also  hastens  the  disintegration  of  the  egg. 
If,  however,  the  eggs  are  treated  with  the  hypertonic  solution 
they  live  and  develop.  This  would  make  it  appear  as  if  the 
membrane  formation  alone  only  hastened  the  natural  death 


Introduction  1,5 


of  the  egg,  while  the  second  factor,  the  treatment  of  the  eg^ 
with  the  hypertonic  sohition,  was  the  Ufe-saving  factor.  Yet 
this  conclusion  is  unwarranted.  It  makes  no  difference  whether 
the  treatment  of  the  unfertilized  egg  with  a  hypertonic  solution 
follows  or  precedes  the  artificial  membrane  formation  (except 
that  the  length  of  time  of  exposure  of  the  eggs  to  the  hyper- 
tonic solution  differs  in  both  cases).  The  writer  found  recent !>• 
that  if  once  the  unfertilized  eggs  have  been  treated  with  a  hyper- 
tonic solution  they  possess  the  second  factor  or  substance 
necessary  for  development,  as  long  as  they  are  alive.  If  at 
any  time  24  or  48  hours  later  they  are  submitted  to  the  process 
of  artificial  membrane  formation  they  will  not  disintegrate, 
but  develop  normally  at  room  temperature.  Now  if  the  treat- 
ment with  the  hypertonic  solution — our  second  factor — were 
really  the  life-saving  agency  in  fertilization  or  artificial  partheno- 
genesis, unfertilized  eggs  which  had  been  treated  with  a  hyper- 
tonic solution  alone  (without  membrane  formation)  should  live 
indefinitely.  This  is,  however,  not  the  case.  If  we  treat  un- 
fertilized eggs  with  a  hypertonic  solution  only,  such  eggs  will 
die  just  as  fast  as  unfertilized  eggs  not  treated  in  this  way. 
If,  however,  such  eggs  are  subsequently  submitted  to  the 
process  of  artificial  membrane  formation  they  will  develop 
and  live  indefinitely.  To  such  eggs  the  artificial  membrane 
formation  becomes  a  life-saving  agency.  From  these  facts 
we  must  conclude  that  both  factors  of  artificial  partheno- 
genesis are  required  to  preserve  the  life  of  the  egg  and  prevent 
its  death. 

This  conclusion  is  supported  by  the  fact  that  for  a  small 
percentage  of  starfish  eggs  or  annelid  eggs  the  mere  act  of  mem- 
brane formation  suflftces  for  development  and  the  prevention  of 
the  death  of  these  eggs.  We  are  forced  to  conclude  that  such 
eggs  contain  or  form  a  substance  which  prevents  the  disintegra- 
tion hastened  as  a  rule  by  the  process  of  membrane  forma- 
tion;  and  which  in  the  sea-urchin  egg  must  be  produced  by  the 


16      Artificial  Parthenogenesis  and  Fertilization 


treatment  with  the  hypertonic  solution  or  with  the  temporary 
suppression  of  oxidations. 

Ten  years  ago  the  writer  found  that  the  hfe  of  the  untreated 
unfertilized  egg  can  be  prolonged  for  some  time  (though  not 
indefinitely)  by  suppressing  the  oxidations  in  the  egg.  This 
indicated  that  the  oxidations  in  the  unfertilized  egg  are  one  of 
the  causes  which  lead  to  the  premature  death  of  the  unfertilized 
egg.  This  conclusion  is  supported  by  the  fact  that  the  mature 
unfertilized  egg  of  the  starfish  dies  much  more  quickly  than  the 
unfertilized  egg  of  the  sea-urchin;  the  rate  of  oxidations  in  the 
mature  but  unfertilized  egg  of  the  starfish  is  comparatively  much 
greater  than  in  the  mature  but  unfertilized  egg  of  the  sea-urchin. 

We  must  therefore  conclude  that  the  oxidations  going  on  in 
the  mature  but  unfertilized  egg  are  one  of  the  causes  that  lead 
directly  or  indirectly  to  its  death;  and  that  in  the  light  of  this 
fact  it  appears  as  if  the  process  of  fertilization  rendered  the  egg 
immune  against  oxidations,  or,  in  other  words,  transformed 
the  egg  from  an  anaerobe  into  an  aerobe. 

10.  Since  physiologists  who  are  not  familiar  with  the  litera- 
ture often  state  that  artificial  parthenogenesis  does  not  lead  to 
the  production  of  larvae  capable  of  development,  it  might  be 
well  to  point  out  that  such  statements  are  contrary  to  fact. 
Delage  has  raised  two  parthenogenetic  larvae  of  the  sea-urchin 
during  sixteen  months  to  the  stage  of  sexual  maturity.^  Both 
were  males.  Loeb  and  Bancroft  raised  a  parthenogenetic  frog 
through  metamorphosis  and  found  that  its  sex  glands  con- 
tained eggs. 2  If  the  raising  of  larvae  were  not  such  a  tedious 
process,  parthenogenetic  animals  would  exist  today  in  large 
numbers,  since  parthenogenetic  larvae  may  be  normal  and 
apparently  healthy. 

1  Delage,  Compt.  rend.  Acad.  d.  Sc,  CXLVIII,  453,  1909. 

2  Loeb  and  Bancroft,  Jour.  Exper.  Zool.,  XIV,  275,  1913. 


II 

SOME  REMARKS  ON  THE  MORPHOLOGY  OF 

DEVELOPMENT 

Since  this  book  is  intended  not  only  for  the  zoolojj;ist,  hut 
more  especially  for  the  physiologist,  pathologist,  and  chemist,  it 
is  necessary  to  give  a  sketch  of  the  development  of  the  animal 
egg.  As  an  example  we  will  use  the  egg  of  the  sea-urchin,  upon 
which  the  majority  of  experiments  on  the  chemical  activation 


P      C7 


Fig.  1. — Unfertilized  egg  of 
the  sea-urchin,  S.  pur  pur  at  us, 
surrounded  by  spermatozoa. 


Fig.  2. — The  same  egg  about 
two  minutes  later,  after  the  en- 
trance of  the  spermatozoon  and 
the  formation  of  the  fertilization 
membrane. 


of  development  have  been  performed.  The  reason  for  this 
is  to  be  found  in  the  fact  that  the  eggs  of  the  sea-urchin  can 
usually  be  obtained  in  large  quantities  and  that  they  form 
the  most  suitable  material  for  our  problem. 

Fig.  1  is  a  picture  of  the  unfertilized  egg  surrounded  by 
spermatozoa.  (The  flagella  of  the  spermatozoa  have  been 
omitted  in  the  drawing.)  As  soon  as  a  spermatozoon  has 
entered,  a  very  characteristic  alteration  takes  place  in  the  egg; 
it  becomes  surrounded  by  the  so-called  fertilization  meml)raiie 
(Fig.  2).     The  mechanism  of  this  membrane  formation  can  Ije 

17 


18      Artificial  Parthenogenesis  and  Fertilization 


more  distinctly  followed  in  the  egg  of  Strongylocentrotus  pur- 
puratus  by  lowering  the  temperature  of  the  sea-water;  by  this 
means,  the  process  of  membrane  formation  is  retarded,  and  it 


<■  •■■".■•■»'.■■'■'>■;■■.    -^J 

Fig.  3 


Fig.  4 


Figs.  3  and  4. — Origin  of  the  fertilization  membrane  through  the  formation 
of  small  vesicles  on  the  surface  of  the  egg. 

can  be  observed  in  its  separate  phases.  Since  we  shall  see  later 
that  this  process  of  membrane  formation  is  the  essential  part 
in  the  activation  of  the  egg,  we  will  trace  it  through  its  separate 
stages. 

The  beginning  of  the  process  shows  itself  in  a  roughening 
of  the  hitherto  smooth  surface  of  the  egg  (Fig.  3).  This  is 
due  to  the  formation  of  countless  tiny  vesicles  which  stand 


Fig.  5  Fig.  6  Fig.  7 

Figs.  5,  6,  and  7. — Further  stages  of  the  process  of  membrane  formation. 

out  on  the  surface  of  the  egg.^  These  small  droplets  quickly 
increase  in  size  (through  absorption  of  sea-water)  and  flow 
together  into  larger  drops  (Fig.  4).     This  goes  on  (Figs.  5,  6,  7) 


Hole. 


1 1  have  never  observed  this  phenomenon  in  the  eggs  of  Arbacia  in  Woods 


Morphology  of  Development 


19 


until  finally  the  contents  of  all  the  drops  have  run  together 
into  a  continuous  layer  around  the  egg^  (Fig.  2).  Hence  the 
surface  lamellae  of  the  tiny  droplets  form  later  the  fertiHzation 
membrane. 

At  higher  temperatures  the  process  of  memljrane  formation 
in  freshly  removed  eggs  usually  proceeds  so  quickly  that  the 
stages  depicted  in  Figs.  3  to  7  are  not  distinctly  seen  and  the  ('ji;g 
passes  directly  from  the  condition  of  Fig.  1  to  that  of  Fig.  2. 
At  first  the  membrane  adheres  closely  to  the  egg,  and  then  of  a 
sudden  the  space  between  the  cytoplasm  and  the  membrane 
increases  enormously.  In  this  latter  way  the  process  of  mem- 
brane formation  occurs  in  the  egg  of  Arbacia. 

After  the  formation  of  this  fertilization  membrane  a 
second  change  takes  place  in  the  surface  of  the  egg,  inasmuch  as 
a  gelatinous  film  or  membrane  {G.M.  Fig.  8)  gradually  appears 
on  the  surface  of  the  cytoplasm.  This 
gelatinous  film  does  not  form  as  sud- 
denly or  quickly  as  the  fertilization 
membrane,  but  only  begins  to  appear 
after  ten  minutes  or  more.  The  writer 
considers  it  possible  that  the  formation 
of  this  film  depends  upon  processes  of 
oxidations,  since  its  formation  is  de- 
layed if  the  oxidations  in  the  egg  are 
retarded  by  the  presence  of  KCN. 

After  membrane  formation,  the 
chemical  processes  which  underlie  de- 
velopment set  in  in  the  egg.  The  nuclear  material  grows  and 
nuclear  division  occurs;  this  nuclear  division,  or  rather  the 
so-called  nuclear  spindle,  is  visible  in  the  egg  which  we  have 
chosen  for  description,  viz.,  that  of  Strongylocentrotus  purpuratus 
(Fig.  9).  The  spindle  formation  visible  in  this  figure  is  imme- 
diately followed  by  cleavage  or  cell  division,  i.e.,  the  partition 

1  So-called  "perivitelline  space." 


FiQ.  8. — Formation  of  a 
gelatinous  flhn  O.M.  around 
the  protoplasm  of  (lu>  egg. 
about  15  minutes  aftvr  the 
formation  of  the  fertilization 
membrane  B.M. 


20       Artificial  Parthenogenesis  and  Fertilization 


of  the  egg  into  two  spheres  or  cells.  The  various  consecutive 
stages  of  this  process  are  depicted  in  Figs.  10  to  13.  First  we 
see  that  the  egg  becomes  somewhat  elongated  in  the  direction 

of  the  axis  of  the  spindle  (Figs. 
10,  11).     This  is  probably  due  to 
the  fact  that  the  protoplasm  flows 
to  the  poles  of  the  spindle  and 
away    from  its  equator.      Then 
there  begins  a  cleavage  of  the  pro- 
toplasm in  the  equatorial  plane 
(Fig.  12),  until  the  egg  consists 
of  two  cells,  each  of  which  pos- 
sesses a  nucleus  (Fig.  13).     This 
process  is  called  the   segmenta- 
tion of  the  egg. 
We  will  now  examine  somewhat  more  closely  this  process 
of  cell  division  or  cleavage.     We  know  two  types  of  cell  division ; 
one  corresponds  to  the  type  of  separation  described  here.     The 


Fig.  9. — Nuclear  division  (spindle 
formation)  in  the  egg  of  S.  pur- 
puratus. 


Fig.  10  Kig.  11 

Figs.  10  and  11. — Beginning  of  segmentation  in  the  egg. 

second  occurs  in  plants  and  consists  in  the  formation  of  a  solid 
membrane  at  the  equator  of  the  cell,  without  the  two  cells 
actually  separating.  At  a  certain  stage,  both  types  of  divisions 
are  identical,  for  in  both  certain  materials  are  carried  to  the 


Morphology  of  Development 


21 


equatorial  plane  of  the  cell  during  the  s])in(lle  stage.  In  i)lant.s 
these  materials  consist  of  cellulose  which  hardens  and  forms  a 
membrane  separating  the  two  cells.  In  animals  they  coasist 
of  a  different  substance  which  possibly  forms  soaps  (Quincke's 
albumin  soap?)  at  the  surface  of  the  egg.  These  substances 
(soaps?)  induce  streaming  phenomena,  which  according  to  the 
writer  lead  to  the  division  of  the  egg  into  two  spheres.^ 


Fig.  12  Fig.  V.i 

Figs.  12  and  13. — The  first  cell  division  completed. 

T.  B.  Robertson  has  lately  brought  forward  a  pretty  demon- 
stration of  this  view.^  If  a  drop  of  olive  oil  is  placed  on  the 
surface  of  water  and  upon  one  diameter  of  the  drop  there  is  laid 
a  thread  which  has  been  previously  moistened  with  an  alkaline 
fluid  (e.g.  N/10  NaOH),  the  drop  divides  into  two  drops,  just 
like  the  sea-urchin  egg  in  the  previously  described  figures.  By 
suitably  varying  the  viscosity  of  the  oil  and  other  conditions 
one  can  reproduce  all  the  phases  of  cell  division  which  can  ])e 
observed  at  cleavage  during  the  separation  of  the  cells.  The 
alkah  on  the  thread  forms  a  soap  with  the  oleic  acid,  and  this 
soap  induces  streaming  phenomena  which  lead  to  the  splitting 
of  the  drop  into  two.^     In  discussing  the  possibility  of  a  synthe- 

iLoeb,  Archiv  f.  Entwicklungsmechanik,  I,  4:68-70,  189G;  X.WII.  i:V^.  IWO; 
Butschli.  ibid.,  X,  52.   1900. 

-T.  B.  Robertson,  Archiv  f.  Entwickhingsmechanik,  XXVII.  29.  1909. 
3  Loeb,  The  Dynamics  of  Living  Matter,  New  York.  1906.  pp.  5a-58. 


22      Artificial  Parthenogenesis  and  Fertilization 

sis  of  nucleins  from  lecithin,  I  have  indicated  that  chohn  must  be 
set  free  in  the  hydrolysis  of  lecithin.^  Robertson  assumes  that 
this  chohn,  which  is  an  alkah,  may  be  the  substance  which  serves 
to  form  the  soap,  and  hence  induces  cell  division.^  Obviously 
other  alkalies  may  also  be  concerned.  Perhaps  in  plants  an 
acid  substance — cellulose — is  conveyed  to  the  equatorial  plane, 
and  hence  in  this  case  a  separation  of  the  two  cells  does  not  take 
place,  but  only  the  formation  of  a  solid  separating  membrane. 
This  process  of  cell  division  is  now  repeated  for  each  cell. 
From  the  two-cell  stage  (Fig.  13)  the  egg  goes  naturally  to  the 
four-cell  stage  (Fig.  14),  eight-cell  stage  (Fig.  15),  etc. 


Fig.    14. — Four-cell   stage   of 
the  egg  of  S.  purpuratus. 


Fig.    15. 
egg. 


-Eight-cell  stage  of  the 


From  the  early  stages  there  is  a  marked  tendency  for  the 
single  cells  to  creep  to  the  surface  of  the  egg;  this  may  depend 
upon  a  tropism,  perhaps  a  positive  chemotropism  of  the  cells 
toward  oxygen.  Owing  to  this  creeping  of  the  cells  to  the 
surface  the  first  larval  stage  of  the  sea-urchin  is  a  hollow  sphere, 
the  so-called  blastula  (Fig.  16).  In  this  stage  there  appear  on 
the  outer  surface  of  the  cells  cilia  (which  are  omitted  in  Fig.  16) 

1  Loeb,  Ueber  den  chemischen  Charakter  des  Befruchtungsvorgangs,  und  seine 
Bedeutung  fur  die  Theorie  der  Lebenserscheinungen,  Leipzig,  1908. 

2  The  interpretation  of  Robertson's  experiment  was  combated  by  McClendon, 
Am.    Jour.    Physiol.,    XXVII,    240,    1910,    and     Archiv  f.    Entwicklungsmechanik, 

XXXIV,  263,  1912.      See  also  T.  B.  Robertson,  Archiv  f.  Entwicklungsmechanik, 

XXXV.  692,  1913. 


Morphology  of  Development 


23 


by  means  of  which  the  blastula  swims  about.  At  this  period, 
the  membrane  is  burst  by  some  unknown  influence,  and  the 
blastula,  which  at  first  swam  around  within  the  egg  sheath 


Fig    16. — Early  blastula  stage  of 
the  sea-urchin  egg. 


Fig.   17. — Gastrula   stage   of 
sea-urchin  egg. 


the 


(fertilization  membrane),  now  moves  about  freely  in  the  water. 
The  larva  very  soon  rises  to  the  surface.  The  next  step  in  the 
development  is  the  gastrula  stage 
(Fig.  17).  On  one  side  the  cells 
grow  into  the  blastocele  and  this  sac 
or  tube  growing  into  the  cavity  is 
the  rudiment  of  the  gut.  On  each 
side  of  the  gut  can  be  seen  indicated 
the  rudiment  of  the  skeleton  in  the 
form  of  two  crystals  or  triasters. 
The  further  developmental  stages 
consist  in  the  organization  of  the  gut 
into  further  divisions,  and  the  out- 
growth of  the  triasters  into  a  larger 
skeleton.  The  originally  spherical 
larva  at  the  same  time  assumes  a 
pyramidal  form.     It  is  called  a  pluteus  (Fig.  18). 

If  the  larvae  are  not  fed,  they  live  some  14  to  18  days  at  a 
temperature  of  about  15°.     The  further  rearing  is  ver}^  tedious 


Fig.  18. — The  pluteus  stage  of 
the  larva  of  S.  purpuratus. 


24      Artificial  Parthenogenesis  and  Fertilization 


and  difficult,  as  the  right  food  of  the  larvae  must  be  raised  in 
the  form  of  cultures.  For  our  experiments  we  shall  consider 
only  the  rearing  of  the  pluteus  stage.  The  kind  of  chemical 
activation  of  the  egg,  called  in  this  book  ''improved  method," 
causes  a  development  in  the  unfertilized  sea-urchin  egg  which  in 
a  great  number  of  cases  corresponds  to  the  form  of  development 
of  the  fertilized  egg  just  described.  The  first  division  alone 
usually  shows  small  irregularities,  but  these  disappear  in  later 
divisions.  On  the  whole,  the  reader  can  also  apply  the  fore- 
going descriptions  to  artificial  parthenogenesis. 

The  experiments  described  in  this  book  were  performed  upon 
eggs  such  as  are  deposited  by  the  female  in  the  water.  Usually 
males  and  females  of  these  forms  are  found  together  in  great 
numbers,  and  on  certain  days  both  sexes  in  one  region  simul- 
taneously pour  their  sexual  cells  into  the  ocean.  On  the  days 
on  which  a  widely  spread  form  spawns,  the  sea  resembles  a  sus- 
pension of  spermatozoa.  The  enormous  numerical  superiority 
of  the  spermatozoa  over  the  eggs  insures  the  fertilization  of 
each  egg.  The  view  that  the  spermatozoon  is  chemotactically 
attracted  by  the  egg  apparently  does  not  hold  for  the  eggs 
of  animals,  although  from  time  to  time  statements  to  the  con- 
trary have  been  published. 


H.  C.  State  College 


Ill 

FERTILIZATION  AND  OXIDATION 

1.  Eighteen  years  ago  the  writer  showed  that  if  freshly 
fertihzed  animal  eggs  (of  sea-urchins  and  fishes)  are  deprived 
of  all  oxygen,  no  nuclear  or  cell  division  is  possible.^  Godlew- 
ski^  and  Samassa^  have  found  the  same  with  frog  eggs,  and 
since  then  I  have  convinced  myself  that  it  holds  for  the  eggs 
of  the  starfish,  annelids,  molluscs,  and  probably  generally.  In 
the  same  way  oxygen  is  necessary  for  the  maturation  of  the  egg. 

In  these  experiments  on  the  prevention  of  development  of 
the  egg  by  lack  of  oxygen,  one  must  guard  against  a  source 
of  error  which  lies  in  the  fact  that  some  time  must  elapse  before 
the  air  or  oxygen  has  been  driven  out  of  the  dish  containing  the 
egg.  Since  in  many  forms  the  first  division  of  the  nucleus 
takes  place  in  a  little  less  than  an  hour,  if  the  temperature  is 
high  enough,  and  since  it  often  requires  as  long,  or  even  a  longer 
period,  to  replace  all  the  oxygen  by  hydrogen,  it  may  easily 
happen  that  a  nuclear,  or  even  a  cell  division,  may  take  place 
after  the  egg  has  been  placed  in  the  stream  of  hydrogen.*  I 
avoided  this  source  of  error  by  cooling  with  ice  the  vessel  con- 
taining the  eggs  to  0°  for  as  long  as  appeared  necessary  from  the 
foregoing  experiments  to  replace  all  the  oxygen  by  hydrogen. 

1  Loeb,  "  Die  physiologische  Wirkiing  des  Sauerstoffmangels,"  Pflilgers  Archiv, 
LXIl,  249,  1895. 

-  Godlewski,  "  Die  Einwirkung  des  Sauerstoflfs  auf  die  Entwicklung  von  Rana, 
etc.,"  Archiv  f.  Entwicklungsmechanik,  XI,  585,  1901. 

3  Samassa,  Verhandl.  d.  naturh.-med.  Vereins  zu  Heidelberg,  IV,  1898,  and  Ver- 
handl.  d.  deutsch.  Zool.  Gesellsch.,  1896  (quoted  from  Godlewski). 

*  It  must  not  be  taken  for  granted  in  general  that  oxygen  is  unimportant,  if 
the  experimenter  does  not  succeed  in  suppressing  all  signs  of  life  after  a  short  pas- 
sage of  hydrogen  or  nitrogen  through  a  vessel.  It  must  not  be  forgotten  that  it 
takes  a  long  time  before  the  last  trace  of  oxygen  is  driven  out,  and  that  often  a 
trace  of  oxygen  is  quite  sufficient  to  render  possible  the  life  phenomenon  under 
investigation. 

25 


26      Artificial  Parthenogenesis  and  Fertilization 


At  this  temperature  the  velocity  of  the  chemical  reactions  in 
the  egg  is  reduced  almost  to  zero.  On  raising  the  temperature 
again,  no  division  of  the  fertilized  egg  takes  place  in  the  hydro- 
gen atmosphere;  but  on  allowing  air  to  enter  the  receptacle,  the 
processes  of  nuclear  and  cell  divisions  are  again  at  once  resumed. 
Perhaps  it  can  be  still  more  strikingly  demonstrated  that  oxida- 
tion is  necessary  for  the  development  of  the  egg,  by  suppressing 
the  oxidation  processes  by  certain  poisons.  It  has  long  been 
known  that  oxidations  in  the  cell  can  be  prevented  by  the  addi- 
tion of  a  little  potassium  cyanide,  even  when  ox3^gen  is  present. 
I  have  found  that  the  addition  of  0.5  c.c.  of  a  1/20  per  cent 
KCN  solution  to  50  c.c.  of  sea-water  is  sufficient  to  stop  almost 
immediately  the  effect  of  the  spermatozoon  in  the  fertilized 
sea-urchin  egg.  When,  however,  such  an  egg  has  been  trans- 
ferred to  normal  sea-water  and  good  aeration  has  been  estab- 
lished, its  development  proceeds  normally,  provided  that  the 
eggs  have  not  remained  too  long  in  the  potassium  cyanide 
solution. 

From  these  facts  the  writer  came  to  the  conclusion  that  one 
essential  effect  of  the  entrance  of  the  spermatozoon  into  the  egg 
of  the  sea-urchin  is  the  acceleration  of  processes  of  oxidation. ^ 
Oxidations  go  on  also  in  the  unfertilized  egg.  He  concluded 
this  from  the  fact  that  the  unfertilized  eggs  in  general  disin- 
tegrate in  a  comparatively  short  time,  while  the  addition  of 
KCN  or  the  withdrawal  of  oxygen  prevented  this  disintegration, 
and  he  explained  this  phenomenon  on  the  assumption  that  the 
oxidations  in  the  unfertilized  eggs  accelerated  their  disintegra- 
tion. The  correctness  of  these  ideas  has  since  been  proved  by 
direct  measurements. 

O.  Warburg  was  the  first  to  measure  the  consumption  of 
oxygen  in  the  unfertilized  and  fertihzed  sea-urchin  egg,  Arbacia 

1  Loeb,  "Ueber  den  chemischen  Charakter  des  Befruchtungsvorgangs," 
Biochem.  Zeitschr.,  I,  183,  1906,  and  also  preface  to  Untersuchunuen  tieber  kunstliche 
Parthenogenese,  Leipzig,  1906. 

2  Loeb,  Pfliiger's  Archiv,  XCIII,  59,  1902. 


2 


Fertilization  and  Oxidation  27 


pustulosa,  at  Naples,  by  Winkler's  method,  and  found  that  after 
fertiUzation  the  egg  consumes  from  six  to  seven  times  as  much 
oxygen  as  before  fertihzation.^  Wastenej^s  and  I  determined 
the  consumption  of  oxygen  in  unfertihzed  eggs  of  Arbada 
at  Woods  Hole.  We  found  that  immediately  after  fertili- 
zation the  egg  consumes  almost  four  times  as  much  oxygen 
as  before  fertilization.^  In  experiments  on  Strongylocentrotus 
purpiiratus  in  Pacific  Grove  we  found  that  immediately  after 
fertilization  the  egg  consumed  five  to  seven  times  as  much 
oxygen  as  before  fertilization.^ 

This  difference  in  the  rate  of  oxidations  in  the  unfertilized 
and  fertilized  eggs  of  the  sea-urchin  is  probably  due  to  the  fact 
that  the  unfertilized  egg  of  the  sea-urchin  is  usually  in  the 
resting  stage,  i.e.,  no  nuclear  divisions  are  going  on  in  it, 
when  it  is  taken  out  of  the  ovary  and  it  generally  remains  in 
this  state  if  no  spermatozoon  enters. 

2.  The  conditions  are,  however,  entirely  different  in  the 
starfish  egg.  This  egg  is,  as  a  rule,  immature  when  taken  out 
of  the  ovary,  but  as  soon  as  it  gets  into  the  sea-water,  it 
may  become  mature;  i.e.,  two  nuclear  divisions  take  place  in 
succession  and  the  polar  bodies  are  thrown  out.  I  have  shown 
in  a  former  paper  that  this  process  requires  the  presence  of  free 
oxygen  in  the  same  way  as  the  developing  sea-urchin  egg.  Lack 
of  oxygen  or  presence  of  KCN  prevents  these  nuclear  maturation 
divisions  in  the  starfish  egg  with  the  same  certainty  as  it  does 
the  nuclear  divisions  in  the  fertilized  sea-urchin  egg.^  And, 
moreover,  I  was  able  to  show  that  a  slightly  alkaline  reaction 
of  the  surrounding  solution  is  as  favorable  to  the  process  of 
maturation  of  the  starfish  egg  as  it  is  to  the  segmentation  of 
the  fertilized  egg  of  Strongylocentrotus  purpiiratus. 

1  O.  Warburg,   Zeitschr.  f.   physiol.   Chem.,  LVII,  6.   1908. 
-  Loeb  and  Wasteneys,  Biochem.  Zeitschr.,  XXXVI,  351,  1911. 
'  Loeb  and  Wasteneys,  Jour.  Biol.  Chem.,  XIV,  469,  1913. 
«  Loeb,  "Maturation,   Natural  Death  and  the  Prolongation  of  the  Life  of 
Unfertihzed  Starfish  Eggs,"   Biol.  Bull.,  Ill,  295,  1902. 


28      Artificial  Parthenogenesis  and  Fertilization 


All  these  observations  point  to  the  conclusion  that  the 
processes  determining  or  underlying  nuclear  division  depend 
upon  oxidations. 

The  eggs  of  the  starfish  must  be  fertilized  at  the  time  the 
second  polar  bod}^  is  given  off  or  immediately  afterward,  since 
otherwise  they  disintegrate  and  cannot  be  fertilized  at  all. 
When  we  compare  the  rate  of  oxidations  in  starfish  eggs  imme- 
diately before  and  after  fertilization,  we  find  no  difference,  as 
Table  I  shows.  It  should  be  remarked  that  while  as  a  rule  100 
per  cent  of  the  sea-urchin  eggs  can  be  fertilized  by  sperm,  in  the 
case  of  starfish  eggs  a  much  smaller  percentage  is  usually  ferti- 
lized since  in  most  cases  not  all  the  eggs  become  mature 
simultaneously. 

TABLE  Ji 


Number  of  Experiment 

Consumption  of 

Oxygen  of  the 
Unfertilized  Eggs 

Consumption  of 

Oxygen  of  the 

Fertihzed  Eggs 

Percentage  of 
Fertilized  Eggs 

I 

0.67  mg. 

0.64 

0.78 

0.34 

0.26 

0.51  mg. 

0.52 

0.85 

0.31 

0.33 

11 

II 

20 

Ill 

25 

IV 

71 

V 

52 

It  is  obvious  that  no  noticeable  increase  in  the  rate  of  the 
oxidation  is  caused  in  this  egg  through  the  entrance  of  the 
spermatozoon.  This  is  intelligible  from  the  fact  that  those 
oxidations  which  lead  to  nuclear  division  were  already  going  on 
in  the  eggs  at  the  time  the  spermatozoon  entered. 

3.  Warburg-  compared  the  rates  of  oxidations  in  the  eight- 
cell  stage  and  the  thirty-two-cell  stage.     Their  ratio  was  as 
4-2  to  6-8;    a  slight  increase.     Wasteneys  and  P  measured 
the  change  in  the  rate  of  oxidations  each  hour  for  five  consecu- 
tive hours  after  fertilization  in  the  eggs  of  Arbacia  at  Woods 

1  Loeb  and  Wasteneys,  Archiv  f.  Entwicklungsmechanih,  XXXV,    556,    1912. 

2  Warburg,  Zeitschr.f.  physiol.  Chem.,  LVII,  1,  1908. 

1  Loeb  and  Wasteneys.  Biochem.  Zeitschr.,  XXXVI,  .351,  1911. 


Fertilization  and  Oxidation  29 


Hole.     The  same  eggs  served  as  material  for  these  determi- 
nations.    Temperature,  23°  C. 

Consumption  of  oxygen  per  hour  before  fertilization  0.24  mg. 

1st  hour  after  fertilization  0.94  mg. 
2d  hour  after  fertilization  0.80  mg. 
3d  hour  after  fertihzation  0.87  mg. 
4th  hour  after  fertilization  0.91  mg. 
5th  hour  after    fertilization  1 .05  mg. 

The  value  for  the  hour  following  fertilization  is  probably 
a  little  too  high  on  account  of  the  presence  of  sperm,  which  was 
washed  away  after  the  first  determination.  That  this  assump- 
tion is  correct  is  shown  by  a  repetition  of  the  experiment. 

Consumption  of  oxygen  per  U  hours  in  fertilized  egg — 

first    U  hours  0.67  mg. 

second  1|  hours  0.74  mg. 

third  U  hours  0.83  mg. 

The  consumption  of  oxygen  increased  about  24  per  cent  in 
4 J  hours.  During  this  same  time  the  eggs  developed  from  the 
one-cell  stage  to  the  thirty-two-cell  stage  or  beyond. 

The  question  arises,  What  causes  this  increase  ?  The  ques- 
tion cannot  be  answered  definitely.  During  the  division  the 
total  surface  of  the  egg  increases.  But  it  must  be  remembered 
that  the  blastomeres  are  in  close  contact  with  each  other  and 
that  hence  only  a  fraction  of  their  surface  is  exposed  to  the 
surrounding  medium.  If  the  intensity  of  oxidations  increases 
with  the  total  free  surface  of  the  eggs,  the  slight  increase  of  the 
rate  of  oxidations  with  the  process  of  segmentation  might  be 

intelligible. 

But  this  is  not  the  only  possibility.  It  had  been  stated  by 
Boveri  that  in  each  cell  division  the  mass  of  the  nucleus  doubled, 
so  that  in  the  eight-cell  stage  the  total  nuclear  mass  would  be 
eight  times  as  great  as  in  the  one-cell  stage.  But  this  has  been 
denied  by  more  recent  workers  like  Miss  Erdmann  and  Conklin.^ 

1  Conklin,  "Cell  Size  and  Nuclear  Size,"  Jour.  Exper.  ZooL,  XII,  1,  1912. 


30      Artificial  Parthenogenesis  and  Fertilization 


According  to  Conklin,  the  average  nuclear  growth  during  cleav- 
age is  not  more  than  5  per  cent  to  9  per  cent  for  each  division  up 
to  the  thirty-two-cell  stage.  The  possibility  remains  that  the 
slight  increase  in  the  rate  of  oxidations  wdth  progressive  cell 
divisions  is  due  to  the  increase  of  the  mass  of  the  nuclei.  This 
would  be  intelligible  on  the  assumption  that  the  rate  of  oxida- 
tion  is  in  proportion  to  the  mass  of  the  nuclei.  Such  a  view 
would  harmonize  with  the  suggestion  expressed  formerly  by  the 
writer,  that  the  nucleus  might  be  the  main  (although  not  the 
only)  oxidizing  organ  of  the  cell.^ 

4.  The  observation  that  the  nucleus  of  the  fertilized  egg 
remains  unaltered  during  lack  of  oxygen  or  presence  of  potas- 
sum  cyanide  shows  that  oxidations  are  the  prerequisites  of  the 
mechanical  processes  of  nuclear  and  cell  division.  It  can, 
however,  be  shown  that  aside  from  oxidations  other  chemical 
processes  are  accelerated  in  the  sea-urchin  egg  by  the  process  of 
fertilization.  When  fertilized  eggs  of  Strong ylocentrotus  pur- 
puratus  are  left  in  sea- water  free  from  oxygen  for  twenty- 
four  hours  at  15°  C,  they  will  not  develop  during  that  time, 
but  they  will  begin  to  develop  at  once  if  oxygen  is  admitted.  It 
\vill  be  found,  however,  that  their  development  is  no  longer 
normal,  since  they  form  abnormal  blastulae  and  never  or  rarely 
reach  the  gastrula  stage.  If  unfertilized  eggs  are  kept  for 
twenty-four  hours  without  oxygen  they  remain  perfectly  normal, 
since  upon  addition  of  sperm  they  develop  normally  and  reach 
the  pluteus  stage.  The  result  is  the  same  if  instead  of  with- 
drawing the  oxygen  we  retard  the  oxidations  by  the  addition 
of  KCN. 

Unfertilized  and  fertilized  eggs  of  the  same  female  were 
placed  in  a  bowl  with  50  c.c.  of  sea-water -f  2  c.c.  1/20  per  cent 
KCN  solution. 2    At  different  intervals,  samples  of  these  eggs 

1  Loeb,  Archie  f.  Entwicklungsmechanik,  VIII,  689,  1899.  Some  authors 
attribute  to  me  the  opinion  that  in  the  protoplasm  no  oxidations  occur.  I  have 
never  expressed  such  an  opinion. 

2  Such  a  solution  stops  the  segmentation  in  fertilized  eggs. 


Fertilization  and  Oxidation  31 


were  replaced  in  normal  sea- water.  Sperm  was  added  to  the 
unfertilized  eggs  after  their  transference  to  ordinary  sea-water. 
The  unfertilized  eggs,  which  had  been  in  the  sea-water  contain- 
ing potassium  cyanide  for  two  days,  developed  in  quite  a  normal 
manner,  whereas  the  eggs  which  had  been  fertilized  before  they 
were  put  into  the  cyanide  sea-water  were  no  longer  able  to 
develop  beyond  the  blastula  stage  after  an  exposure  of  only 
twenty-four  hours  to  this  solution.  An  exposure  of  five  hours' 
duration  to  the  cyanide  sea-water  was  already  harmful  to  the 
fertilized  eggs;  this  was  shown  by  the  fact  that  while  such 
eggs  did  develop  after  transference  to  ordinary  sea-water, 
many  larvae  died  during  the  first  two  days.^ 

These  experiments  leave  no  doubt  that  fertilization  gives  rise 
to  a  class  of  chemical  reactions  in  the  egg  which  can  proceed 
independently  of  oxidations.  It  is  very  likely  that  other 
reactions  besides  oxidations,  e.g.,  hydrolyses,  take  place  in  the 
egg.  If  such  hydrolyses  lead  to  the  formation  of  harmful 
oxidizable  substances,  e.g.,  lactic  acid,  it  can  be  understood 
why  lack  of  oxygen  must  in  time  lead  to  the  death  of  the 
fertilized  egg;  without  oxygen  the  harmful  substances,  which 
are  quickly  rendered  harmless  by  oxidation  (or  converted  into 
substances  like  CO2,  which  can  be^hminated),  can  now  accumu- 
late in  the  cell.  Assuming  that  such  hydrolyses  are  set  up  in 
the  egg  by  fertilization,  while  they  are  lacking  or  are  very  slow 
in  the  unfertilized  egg,  we  could  understand  why  the  fertilized 
egg  suffers  more  quickly  than  the  unfertilized  egg  from  lack  of 
ox3^gen  or  from  the  prevention  of  oxidations  by  potassium 
cyanide. 

5.  Wasteneys  and  the  writer  raised  the  question  whether  the 
oxidations  are  the  independent  variable  in  the  development  of 
the  egg.^     From  all  we  know,  we  should  expect  that  hydrolj'ses 

1  Loeb,  "Versuche  ueber  den  chemischen  Charakter  des  Befruchtungsvor- 
gangs,"  Biochem.  Zeitschr.,  I,  189,  1906. 

2  Loeb  and  W^asteneys,  "Sind  die  Oxydationsvorgange  die  unabhiingige 
Variable  in  den  Lebensersclieinungen  ?  "  Biochem.  Zeitschr.,  XXXVI,  345-56,  1911. 


32      Artificial  Parthenogenesis  and  Fertilization 


are  the  independent  variable,  and  that  the  oxidations  are  deter- 
mined or  regulated  by  the  hydrolytic  processes.  Our  investi- 
gations do  not  contradict  such  a  view.  Since  we  are  not  able 
to  measure  the  hydrolytic  processes  in  the  egg  directly,  we  tried 
to  solve  our  problem  with  the  aid  of  the  temperature  coefficient. 
We  determined  the  temperature  coefficient  for  the  velocity  of 
segmentation  in  the  egg  of  Arhacia  by  measuring  the  time 
which  elapses  from  the  moment  of  fertilization  to  the  moment 
of  the  division  of  the  egg  into  two  cells  for  various  temperatures. 
Then  we  measured  the  influence  of  the  same  variation  of  tem- 
perature upon  the  rate  of  oxidations  in  the  cell.  If  the  oxida- 
tions were  the  independent  variable  for  the  development  of  the 
egg  the  temperature  coefficients  for  both  processes  should  be 
identical  or  run  parallel.  This  is,  however,  not  the  case.  The 
experiments  were  made  on  the  eggs  of  Arhacia  at  Woods  Hole. 
The  time  which  elapsed  between  fertilization  and  the  first 
segmentation  was  as  follows : 

TABLE  II 


Temp.  Deg.  C. 


7., 
8.. 
9., 
10.. 
12., 
15., 
16., 

m 


18, 


Time  Required  for 

First  Segmentation 

Minutes 


498 

410 

308 

217 

4   +143* 

3^+  96^ 

2  +  831 

2^+68 

68 


Temp.  Deg.  C. 


20. 
22. 
25. 
26. 
271 
30. 
31. 
32. 


35 


Time  Required  for 

First  Segmentation 

Minutes 


56 

4   +43 
3^+36^ 
2  +31^ 

2i+3U 

33 

37 

No  segmentation; 
eggs  suffered 


*  Where  the  times  are  given  in  the  form  of  a  sum  the  eggs  were  put  into  the 
thermostat  as  many  minutes  after  fertilization  as  the  first  figiu"e  indicates. 

The  temperature  coefficients  for  segmentation  are  the 
greater  the  lower  the  temperature.  For  the  interval  7°-17° 
the  coefficient  is  more  than  three  times  as  large  as  for  the  interval 
17.5°-27.5°  (Table  III). 


Fertilization  and  Oxidation 


33 


TABLE  III 


Interval  of 
Temperature 

Temperature  Co- 
efficient for  10°C. 
for  the  Velocity  of 
Segmentation  in 
the  Egg  of  Arbacia 

Interval  of 
Temperature 

Temperature  Co- 
efficient for  10°C. 
for  the  Velocity  of 
Segmentation  in 
the  Egg  ot  Arbacia 

7°-1 7° 

7.3 

6.0 

>4.0 

~3.9 

3.3 

15°-25° 

2.6 

S°-l  8° 

16°-26° 

2.6 

9°-19° 

17.5°-27.5° 

20°-30° 

2.2 

1 0°-20° 

1.7 

12°-22° 

A  determination  of  the  temperature  coefficient  of  oxida- 
tions in  the  egg  of  Arbacia  did  not  show  such  a  variation. 


TABLE  IV 


Interval  of 
Temperature 

Temperature  Co- 
efficient for  10° 
for  the  Velocity  of 
Oxidations 

Interval  of 
Temperature 

Temperature  Co- 
efficient for  10° 
for  the  Velocity  of 
Oxidations 

oo      100 

2.18 
2.16 
2.00 
2.17 
2.45 

15°-25° 

2.24 

'^°   1  ^° 

17°-27° 

2.00 

')        1.0 •  • 

7°    17° 

20°-30° 

1.96 

10°   '>0° 

22°-32° 

1.40 

XL»       ajU     ......... 

13°-23"    

^ 

The  temperature  coefficients  of  oxidations  for  a  difference 
of  10°  C.  are  practically  identical  throughout.  For  the  interval 
7°-17°  the  coefficient  is  2.0  and  for  17°-27°  it  is  also  2.0.  This 
result  points  toward  the  probability  that  the  rate  of  cell  division 
is  not  directly  determined  by  the  rate  of  oxidations,  although 
the  possibility  exists  that  besides  the  influence  of  temperature 
upon  the  velocity  of  chemical  reactions  an  influence  upon  some 
physical  property  of  the  egg  is  superposed  (e.g.,  viscosity) 
which  makes  itself  felt  only  or  chiefly  at  the  lower  temperatures 
and  becomes  the  stronger  the  lower  the  temperature. 

6.  The  writer  has  shown  that  segmentation  and  develop- 
ment can  go  on  only  if  the  concentration  of  the  HO  ions  in  the 
surrounding  solution  reaches  a  certain  height;    this  height  is 


34      Artificial  Parthenogenesis  and  Fertilization 


greater  for  the  egg  of  Strongylocentrotus  purpuratus  than  for 
Arbacia. 

vSea-water  is,  according  to  van't  Hoff,  a  mixture  of  the 
following  composition :  100  molecules  NaCl,  2 . 2  molecules  KCl, 
1.5  molecules  CaClg,  7.8  molecules  MgClg,  and  3.8  molecules 
MgS04.'  To  this,  traces  of  NaHCOs  and  Na2HP04  are  to  be 
added.  The  osmotic  pressure  of  the  ocean  water  show^s  local 
variations.  The  eggs  of  the  animals  in  Pacific  Grove  develop 
best  if  the  salts  are  used  in  a  half  grammolecular  concentration. 
For  the  fauna  in  Woods  Hole,  on  the  Atlantic,  a  slightly  higher 
concentration,  about  21/40  m  is,  perhaps,  the  optimum,  al- 
though m/2  solutions  give  almost  the  same  result.  Solutions 
of  25/40  m  are  decidedly  injurious. 

The  reaction  of  the  sea-water  is  slightly  alkaline.  In 
Pacific  Grove  the  concentration  of  the  free  HO  ions  of  the 
sea-water  seems  to  lie  between  10"^  and  10~^  N,  since  it  is 
alkaline  to  neutral  red  but  not  to  phenolphthalein.  The 
concentration  of  the  HO  ions  in  the  sea-water  at  Woods  Hole 
is  slightly  higher  and  may  reach  10"^  N. 

If  w^e  make  m/2  solutions  containing  NaCl,  KCl,  CaCl2, 
MgCU,  and  MgS04  in  the  right  proportion,  the  newly  fertilized 
eggs  of  Strongylocentrotus  will,  as  a  rule,  not  be  able  to  develop 
to  the  larval  stage  in  such  a  solution,  unless  the  ^no  is  above 
10"^  N.  The  eggs  of  various  females  differ  slightly  in  their 
minimum  Chq.  It  is,  of  course,  necessary  to  free  the  eggs 
carefully  from  all  traces  of  sea-w^ater  by  washing  them  re- 
peatedly in  neutral  solutions  before  submitting  them  to  the 
experiment. 

A  van't  Hoff  solution  w^as  prepared.  The  Chq  was  about 
10~"  N,  i.e.,  the  solution  was  neutral.  To  50  c.c.  of  this  solution 
w^ere  added  0,  0.1,  0.2,  0.4,  and  0.8  N/100  KOH.  Newly 
fertilized  eggs  of  Strongylocentrotus  purpuratus  were  put  into 
these   solutions.     In  the   neutral   solution   no   egg  developed 

1  We  will  call  this  solution  for  the  sake  of  brevity  the  van't  HoflF  solution. 


Fertilization  and  Oxidation  35 


bej^ond  the  four-  to  eight-cell  stage.  Addition  of  0 . 1  c.c.  N/100 
KOH  allowed  a  few  eggs  to  reach  the  blastula  stage;  addition 
of  0.2  c.c.  N/100  KOH  allowed  60  per  cent  to  reach  the  blastula 
stage  and  with  0.4  and  0.8  c.c.  N/100  KOH  all  the  eggs 
developed  into  larvae. 

If  we  add  more  KOH  to  50  c.c.  of  the  van't  Hoff  solution, 
we  find  that  the  addition  of  0.4  c.c.  N/10  KOH  interferes 
already  with  their  development;  if  we  add  0.8  c.c.  N/10  KOH, 
or  more,  to  50  c.c.  van't  Hoff  solution,  no  egg  can  segment.^ 

If  one  wishes  to  obtain  the  best  type  of  larvae  it  is  better 
to  add  varying  concentrations  of  NaHCOg.  If  to  50  c.c.  of  the 
neutral  van't  Hoff  solution  are  added  0,  0. 1,  0.2,  0.4,  0.8,  1 .0, 
2.0  c.c.  m/20  solution  of  NaHCOg;  in  the  solution  without  and 
with  only  0 . 1  c.c.  NaHCOg  only  early  segmentations  take  place; 
in  all  the  other  solutions  the  eggs  will  develop  into  larvae.  In 
the  solution  with  1.0  c.c.  or  more  NaHCOg  the  eggs  already 
suffer. 

It  seems  that  the  faintly  alkaline  solution  is  chiefly  necessary 
only  for  the  first  development  of  the  eggs  of  purpuratus.  Later 
they  are  able  to  develop  in  a  neutral  but  not  in  an  acid  solution. 
The  lowest  ^ho  for  the  development  of  the  egg  of  Arbada  at 
Woods  Hole  is  about  10'^°  N,  i.e.,  these  eggs  can  begin  to 
develop  not  only  in  a  neutral  but  even  in  a  faintly  acid  solu- 
tion. This  difference  between  the  two  kinds  of  eggs  may 
be  of  some  importance  in  regard  to  the  difference  in  their 
response  to  the  agencies  of  artificial  parthenogenesis,  as  we 
shall  see  later. 

The  writer  published  years  ago  a  paper  in  which  he  showed 
that  the  development  of  the  eggs  of  Arhacia  is  retarded  and 
finally  inhibited  if  increasing  quantities  of  acid  are  added  to 
the  sea-water.  He  has  since  vainly  attempted  to  show  that 
the  rate  of  development  of  the  sea-urchin  egg  can  be  increased 
with  the  increase  of  the  concentration  of  hydroxylions  in  the 

1  Loeb,  Biochem.  Zeitschr.,  II,  88,  1906. 


36      Artificial  Parthenogenesis  and  Fertilization 

sea-water.  This  leads  him  to  beUeve  that  these  eggs  develop 
best  in  a  solution  in  which  the  concentration  of  hydroxjdions 
equals  that  of  the  sea-water;  and  that  while  it  is  possible  to 
delay  their  development  by  a  lowering  of  this  concentration, 
no  acceleration  can  be  produced  if  the  ^oh  in  the  sea-water 
is  raised.  This  statement  is  corroborated  by  the  fact  referred 
to  above  that  the  addition  of  some  NaHCOs  is  more  favor- 
able for  the  development  of  the  larvae  than  the  addition  of 
NaOH. 

7.  Warburg  states  that  it  is  possible  to  raise  the  rate  of 
oxidations  in  the  fertilized  egg  through  the  addition  of  NaOH, 
but  not  by  the  addition  of  NH4OH.  Since  he  was  able  to  show 
that  NH4OH  diffuses  into  the  cell  while  NaOH  does  not,  he 
concludes  from  this  and  a  similar  observation^  ''that  all  the 
substances  which  increase  the  oxidations  in  the  fertilized  .eggs 
belong  to  that  class  which  according  to  Overton  cannot  enter 
into  the  living  cell"  (p.  328).  ''The  influence  of  an  increase 
of  the  concentration  of  the  HO  ions  upon  respiration  is  neither 
determined  by  the  entrance  of  ions  into  the  eggs,  nor  by  their 
reacting  with  the  membrane  of  the  cytoplasm,  but  merel}' 
by  their  presence  in  the  solution  surrounding  the  egg"  (p.  314). 
Warburg  quotes  determinations  of  the  oxygen  consumption  of 
newly  fertilized  eggs  of  Strongylocentrotus  at  Naples  for  three 
different  concentrations  of  NaOH,  10"^  N,  10"^  N,  and  lO'^  N. 
The  ratio  of  these  oxidations  in  three  solutions  was  1.4:3.9:8.1. 
Only  in  one  of  these  three  solutions  did  the  eggs  develop,  namely, 
in  the  one  with  the  Cqh  10"^  N.  Of  the  two  others  the  one 
was  too  acid,  the  other  too  alkahne.  NH4OH  did  not  raise 
the  rate  of  oxidations  perceptibly. 

1  Warburg,  "Ueber  die  Oxydationen  in  lebenden  Zellen  nach  Versuchen  am 
Seeigelei,"  Zeitschr.f.  physiol.  Chem.,  LXVI,  305,  1910.  The  other  observation 
not  discussed  in  the  text  refers  to  an  increase  of  oxidations  in  the  fertilized  egg 
under  the  influence  of  hypertonic  solution.  Wasteneys  and  I  were  not  able  to 
confirm  this  statement  of  Warburg;  we  found  that  the  oxidations  in  the  fertilized 
egg  of  Strongylocentrotus  purpuratus  are  not  increased  if  the  eggs  are  put  into  a 
hypertonic  solution. 


Fertilization  and  Oxidation 


37 


These  experiments  might  give  the  reader  two  impressions, 
first,  that  the  rate  of  oxidation  increases  steadily  with  the  con- 
centration of  hydroxyhons  or  of  the  NaOH  in  the  surrounding 
sohition;  and,  second,  that  the  normal  oxidations  take  place 
at  the  surface  of  the  egg.  Experiments  made  by  Wasteneys 
and  the  writer,  however,  do  not  warrant  such  conclusions. 
The  experiments  were  made  on  the  eggs  of  Strongylocentrotus 
purpuratus  in  Pacific  Grove  and  consisted  in  a  more  complete 
determination  of  the  effect  of  varying  concentrations  of  NaOH 
and  NH4OH  upon  the  oxidations.^ 


TABLE  V 


Amount  of  NaOH  Added  to 

50  c.c.  m/2  NaCl+KCl+ 

CaCL 


0.0  C.C.  N/10 
0.3  c.c.  N/10 
0.4  c.c.  N/10 
0.5  c.c.  N/10 
0.6  c.c.  N/10 
0.7  c.c.  N/10 
0.8  c.c.  N/10 


NaOH 
NaOH 
NaOH 
NaOH 
NaOH 
NaOH 
NaOH 


Coeffi- 

cient of 

Oxida- 

tions 

1.00 

1.08 

1.08 

1.13 

1.18 

1.17 

1.51(?) 

Amount  of  NaOH  Added  to 

50  c.c.  m/2  NaCl+KCH- 

CaCl. 


0.9  C.C.  N/10  NaOH 

1.0  c.c.  N/10  NaOH 

1.1  c.c.  N/10  NaOH 

1.2  c.c.  N/10  NaOH 

1.3  c.c.  N/10  NaOH 

1.4  c.c.  N/10  NaOH 


Coeffi- 
cient of 
Oxida- 
tions 


1.44 
1.55 
1.87 
1.93 
2.12 

Eggs 
injured 


This  and  similar  experiments  show  very  plainly  that  the 
addition  of  even  0.5  c.c.  or  0.7  c.c.  N/10  NaOH  to  50  c.c. 
(m/2  NaCl+KCl+CaCla)  does  not  raise  the  rate  of  oxidations 
noticeably;  and  that  a  considerable  rise  in  the  rate  of  oxidations 
does  not  take  place  until  more  than  0.8  c.c.  N/IO  NaOH 
has  been  added.  These  concentrations  can  no  longer  be  con- 
sidered normal,  since  an  addition  of  from  0.2  to  0.4  c.c. 
N/10  NaOH  to  50  c.c.  (NaCl+KCl+CaCla)  suffices  already 
to  suppress  the  development  of  the  egg.  It  is  therefore 
obvious  that  the  increase  of  the  rate  of  oxidations  in  the 
unfertilized  egg,  under  the  influence  of  excessive  quanti- 
ties of  NaOH,  cannot  be  utilized  for  any  conclusions  upon  the 

1  Loeb  and  Wasteneys,  Jour.  Biol.  Chem.,  XIV,  459,  1913. 


38      Artificial  Parthenogenesis  and  Fertilization 

normal  oxidations  in  the  sea-urchin  egg.  On  the  contraiy, 
our  experiments  prove  definitel}'  that  low  concentrations  of 
NaOH  have  practically  no  effect  upon  the  rate  of  oxidations. 

All  the  facts  become  intelligible  on  the  assumption  that 
the  increase  in  oxidations  under  the  influence  of  high  concen- 
trations of  NaOH  is  due  to  an  injurious  (a  kind  of  etching?) 
effect  of  the  latter;  this  would  make  it  clear  why  the  weak 
bases,  such  as  NH4OH,  do  not  produce  an  increase  in  the  rate 
of  oxidations  and  why  low  concentrations  of  NaOH  have  no 
effect  either.  The  fact  that  NH4OH  enters  the  egg,  while 
NaOH  does  not,  has  probably  nothing  to  do  with  this  result. 

The  energetics  of  development — the  nature  of  the  sub- 
stances which  undergo  oxidation,  and  the  way  the  energy  is 
utilized — has  been  investigated  by  TangP  and  his  pupils,  by 
Bohr  and  Hasselbalch,^  and  by  Meyerhof.^  Since  the  experi- 
ments on  artificial  parthenogenesis  are  not  so  intimately  con- 
nected with  these  experiments  as  with  those  on  oxidation,  we 
may  be  pardoned  for  not  discussing  them  in  this  book. 

1  Tangl  Pfliigers  Archiv,  XCIII,  327;  XCVIII,  490;  CIV,  024;  CXXI,  437; 
Biochem.  Zeitschr.,  XLIV,  180,  1912. 

^  Skand.   Arch.,  XIV,  398,   1903. 

3  Meyerhof,  Biochem.  Zeitschr.,  XXXV,  246,  1911. 


IV 

HYDROLYTIC  PROCESSES  IN  THE  GERMINATION  OF 

OIL-CONTAINING  SEEDS 

Since  we  mentioned  the  fact  that  in  the  fertihzed  egg  hydro- 
lytic  processes  (or  at  least  reactions  other  than  oxidations) 
take  place,  and  since  little  ^r  nothing  is  known  about  these 
reactions  in  animal  eggs,  a  discussion  of  the  processes  occurring 
in  germinating  oily  seeds  may  be  of  interest.  As  an  example, 
the  investigations  of  Hover  with  Connstein  and  Wartenbcrg 
may  be  used.     The  experiments  were  carried  out  by  Hoyer. 

If  castor  beans  are  ground  up  with  water,  and  the  resulting  seed- 
oil  emulsion  (seed-milk)  is  left  alone  for  a  few  days,  there  is  observed 
after  a  time  a  sudden,  rapid  increase  of  the  mass  of  acid,  whereby  the 
neutral  oil  contained  in  the  castor  bean  is  converted  into  acid  and 
glycerin.  This  obser\'ation,  made  for  the  first  time  some  four  years 
ago,  was  the  starting-point  for  the  investigation  of  the  decomposition 
of  fat  by  means  of  the  lipolytic  ferments  contained  in  plant  seeds,  and 
especially  in  those  of  the  castor  bean.  This  ferment,  which  has  been 
worked  at  ever  since  from  several  sides,  has  been  used  successfully  as  a 
technical  fat-splitting  process. 

On  the  establishment  of  the  conditions  mentioned,  i.e.,  the  grind- 
ing up  of  the  seeds,  the  fat-splitting  effect  of  the  castor  bean  ferment 
does  not  set  in  for  some  time,  but  then  it  occurs  suddenly.  We  recog- 
nized the  reason  for  the  appearance  of  this  sudden  action  in  the  fact 
that  an  intensification  of  the  decomposition  of  the  fat  only  occurs 
when  a  sufficient  mass  of  acid  is  present.  For  the  fat-splitting  effect 
of  the  castor  bean  can  be  at  once  produced,  if  at  the  start  a  certain 
small  quantity  of  acid  or  the  salt  of  an  acid  be  added  to  the  mass. 
This  amount  differs  according  to  the  kind  of  acid.  In  a  later  publica- 
tion of  mine  the  amounts  of  these  acids  have  been  accurately  deter- 
mined; whence  it  appears  that  of  the  acids  examined,  butyric  allows 
the  widest  latitude  with  regard  to  the  quantity  employed,  whilst  sul- 
phuric and  oxalic,  for  example,  require  a  very  close  adherence  to 
their  proportionate  quantities.^ 

1  Hoyer,  "  Ueber  fermentative  Fettspaltung."  Zeitschr.  f.  phusiol.  Chem., 
L,  414,  1907. 

39 


40       Artificial  Parthenogenesis  and  Fertilization 


The  many  researches  of  Hoyer  next  aimed  at  ascertaining 
what  was  the  nature  of  the  acid  which  is  formed  in  the  seed 
itself  and  conditions  the  sudden  increase  in  the  hydrolysis 
of  the  oil  in  the  germinating  seed.  It  is  found  to  depend 
mainly  upon  lactic  and  carbonic  acids,  together  with  a  relatively 
small  amount  of  acetic  and  formic  acids.  There  occur  there- 
fore two  fundamental  processes  in  the  conversion  of  the  oil  in 
germination.  One  process  is  the  fermentative  production  of 
acids:  lactic,  carbonic,  acetic,  and  formic;  the  second  process, 
which  depends  upon  the  first,  is  the  "activation"  of  the  lipolytic 
enzyme  through  the  acid  produced  in  the  seed. 

Investigations  with  the  acids  produced  in  the  seed  itself 
elicited  the  fact  that  these  acids  are  able  to  produce  the  fat- 
splitting  effect  in  the  castor  bean.  One  fact,  which  is  of  bio- 
logical importance,  may  be  mentioned  here.  If  Hoyer  chose 
for  his  investigation  a  castor  bean  that  had  hardly  begun  to 
germinate,  and  cut  it  in  half  (after  washing  it),  it  appeared  that 
the  oil-splitting  enzyme  had  a  weaker  effect  in  the  half  of  the 
seed  which  contained  the  embryo  than  in  the  half  separated 
from  the  embryo.  In  well  germinated  seeds  there  was  prac- 
tically no  more  ferment  present.  "In  the  life  history  of  the 
castor  bean,  the  ferment  becomes  inoperative  after  the  per- 
formance of  its  fat-splitting  function  in  the  same  proportion 
as  the  castor  oil  is  prepared  for  the  growth  of  the  embryo."^ 

These  investigations  give  us  an  idea  of  the  complicated 
character  of  the  processes  in  the  development  of  the  embryo. 
The  taking-up  of  water  leads  to  a  process  of  hydrolysis  in  the 
seed,  of  which  the  end-products  are  certain  acids — lactic  and 
carbonic.  These  acids  serve,  according  to  Hoyer's  description, 
for  the  "activation^'  of  the  lipase  contained  in  the  castor 
bean.  As  in  all  analyses  of  life  phenomena,  we  are  here  too 
dealing  with  a  catenary  series  of  reactions.  It  seems  that  in  the 
sea-urchin  *S.  purpuratus  also  a  sudden  increase  in  the  acid 

1  Hoyer,  Ber.  d.  deutach.  chem.  Ges.,  XXXVII,  1436,  1904. 


Germination  of  Seeds  41 

content  takes  place  upon  fertilization.  When  two  drops  of  a 
1/100  grammolecular  solution  of  neutral  red  are  added  to  50  c.c. 
of  sea-water,  the  sea-water  immediately  assumes  a  yellow  color, 
owing  to  its  alkaline  reaction. 

If  now  unfertilized  and  freshly  fertilized  eggs  of  the  sea-urchin 
Strongylocentrotus  purpuratus  are  placed  at  the  same  time  in  this  solu- 
tion, both  kinds  of  eggs  quickly  take  on  a  red  color.  But  if,  after  20 
to  40  minutes,  they  are  brought  back  into  normal  colorless  sea-water, 
the  unfertilized  eggs  gradual h^  lose  their  color,  while  the  fertilized 
eggs  become  a  still  deeper  red.  One  receives  the  impression  that, 
while  the  unfertilized  eggs  give  up  again  the  coloring  matter  to  the 
surrounding  sea- water,  the  fertilized  eggs  continue  to  take  it  up. 
After  about  an  hour,  therefore,  two  kinds  of  eggs  are  to  be  found  in 
the  bowl  of  sea-water,  some  colorless,  or  very  slightly  colored,  and  others 
colored  a  deep  red;  the  latter  are,  without  exception,  eggs  which  possess 
a  membrane  and,  later  on,  segment — being  therefore  fertilized  eggs, 
while  the  colorless  eggs  possess  no  membrane  and  are  unfertihzed. 
Neutral  red  is  an  alkaline  coloring  matter,  and  therefore  combines 
in  the  egg  with  an  acid.  This  retention  of  neutral  red  indicates  that  it 
is  held  in  closer  combination  in  the  fertilized  than  in  the  unfertilized 
egg,  where  it  can  be  quickly  lost.^ 

The  yolk  of  the  egg  contains  a  relatively  large  amount  of  fat 
and  lipoids.  The  eggs  of  most  invertebrates  are  small,  not  very 
far  from  the  limit  of  vision  of  the  unaided  eye.  Nevertheless 
they  contain  reserve  materials  in  relatively  large  quantities, 
and  among  these  reserve  substances  are  found  fats  or  lipoids. 
Now  it  is  quite  possible,  if  not  probable,  that  the  commence- 
ment of  development  in  plant  embryos  possesses  certain  points 
in  common  with  that  in  the  eggs  of  animals,  viz.,  in  regard  to 
the  role  of  the  decomposition  of  fats  or  lipoids.  But  it  must 
also  be  borne  in  mind  that  the  fats  or  lipoids  require  a  some- 
what different  treatment,  according  as  they  are  the  more  solid 
or  liquid.  In  oil-containing  plant  seeds  we  have  to  deal  mostly 
with  liquid  oils;   in  animals  the  solid  palmitin  and  stearin  fats 

1  Loeb,  "  Weitere  Beobachtungen  ueber  den  Einfluss  der  Befruchtung  uud  der 
Zahl  der  Zellkerne  auf  die  Saurebildung  im  Ei,"  Biochem.  Zeitschr.,  II.  34.  1906. 


42       Artificial  Parthenogenesis  and  Fertilization 


predominate.  One  can  understand  that  for  the  utilization  of 
such  soUd  fats  in  the  egg,  a  process  must  come  into  play,  which 
is  superfluous  in  seeds  that  contain  oil,  on  account  of  the 
original  fluid  state  of  the  oil:  that  process  is  the  liquefaction 
of  the  fat.  We  have  already  mentioned  that  this  circumstance 
can  be  taken  into  consideration  in  the  membrane  formation  of 
eggs.  It  may,  in  a  word,  depend  upon  the  fact  that  at  mem- 
brane formation  the  fats  or  lipoids  are  converted  into  a  form 
in  which  they  can  be  more  easily  decomposed  or  oxidized. 

A  clearer  analogy  between  the  germination  of  the  seed  and 
the  development  of  the  egg  is  the  fact,  already  proved  by 
Moritz  Traube,^  that  the  germination  of  the  seed  too  is  only 
possible  in  the  presence  of  free  oxygen.  In  the  beginning  of 
germination  in  the  seed,  just  as  in  the  beginning  of  develop- 
ment in  the  egg,  we  are  dealing  with  a  causation  of  syntheses 
out  of  the  constituent  parts  of  the  cytoplasm,  and  for  this 
process  free  oxygen  is  necessary;  and  moreover,  nuclear  and 
cell  division  are  also  concerned,  for  which,  likewise,  free  oxygen 
is  necessary. 

1  Moritz  Traube,  Gesammelte  Abhandlungen,  Berlin,  1896,  p.  148. 


SOME  EARLIER  OBSERVATIONS  ON  NATURAL  PAR- 
THENOGENESIS IN  INSECTS 

Long  before  the  importance  of  the  spermatozoon  in  fertiU- 
zation  was  fully  recognized,  Reaumur,  Bonnet,  and  a  number 
of  other  eighteenth-century  authors  had  established  the  fact 
that  plant  lice  bring  forth  living  young  without  previously 
pairing.  Kirby^  found  that  under  ordinary  conditions  of  tem- 
perature and  moisture,  the  parthenogenetic  generations  of 
aphides  can  follow  one  another  for  four  years  (possibly  in- 
definitely) without  as  a  rule  giving  rise  to  males.  Only  under 
special  conditions  do  plant  lice  produce  both  sexes,  which  then 
pair.  This  pairing  leads  to  the  laying  of  eggs  from  which 
hatch,  without  exception,  parthenogenetic  females  that  give 
birth  to  living  young. 

These  observations  led  entomologists  to  search  for  further 
cases  of  parthenogenesis.  It  was  soon  discovered  that  certain 
butterflies,  belonging  to  the  genus  Solenobia,  lay  unfertilized 
eggs^  which  develop  eventually  in  a  thoroughly  normal  manner 
into  butterflies.  Von  Siebold,  who  repeated  and  confirmed 
these  investigations,  also  found  parthenogenetic  reproduction 
in  another  butterfly.  Psyche  helix,^  of  which  the  males  were 
quite  unknown  at  that  time.  In  all  these  cases  not  only  were 
the  larvae  arising  from  the  unfertihzed  eggs  normal,  but  they 
also  developed  into  perfectly  normal  sexually  mature  insects. 

But  the  greatest  sensation  was  aroused  by  the  observations 
of  Dzierzon  on  parthenogenesis  in  bees.'*     He  was  led  to  the 

1  Quoted  after  Ratzeburg,  Die  Forstinsekten,  Part  III,  1844. 

2  We  gather  from  von  Siebold's  monograph  that  these  facts  were  first  dis- 
covered by  DeGeer. 

3  Von  Siebold,  Wahre  Parthenogenese  bei  Schmetterlingen  und  Bienen,  Leipzig, 
1856. 

*  According  to  von  Siebold,  Dzierzon  first  published  his  observations  and  con- 
clusions in  the  year  1845  (in  the  Eichstadter  Bienemeitung). 

43 


44       Artificial  Parthenogenesis  and  Fertilization 

conclusion  that  male  bees — the  drones — arise  from  unfertilized 
eggs,  workers  and  queens,  on  the  other  hand,  from  fertilized 
eggs.  He  discovered  that  the  queen  copulates  only  once  in 
her  life,  and  that  this  never  takes  place  in  the  hive,  but  in  the 
air,  during  the  so-called  nuptial  flight.  After  pairing,  the 
sperm  remains  in  a  vesicle,  the  female's  receptaculum,  the  duct 
of  which  is  passed  by  the  egg.  If,  now,  the  queen  is  laying 
an  egg  in  a  worker  cell,  a  trace  of  sperm  is  pressed  out  of  the 
receptaculum  when  the  egg  passes  the  opening  of  its  duct,  and 
so  the  egg  is  fertilized.  But  when  the  queen  lays  an  egg  in 
the  larger  drone  cell,  the  egg  passes  the  duct  without  any 
sperm  being  pressed  out.  Von  Siebold  represented  that  bees 
behave  consciously;  but  it  is  more  likely  that  a  purely  physio- 
logical explanation  may  be  found.  It  is,  e.g.,  possible  that  in 
the  narrower  worker  cells  the  muscles  which  empty  the  recep- 
taculum are  reflexly  or  mechanically  set  in  activity,  while 
the  mechanical  stimulus  thereto  is  lacking  in  the  wider  drone 
cells.  ^  Dzierzon  was  able  to  bring  to  the  support  of  this  view 
a  series  of  observations;  thus,  for  example,  queens  which 
have  been  prevented  from  taking  the  nuptial  flight  by  defective 
wing  development  invariably  give  rise  to  drones;  the  same  is 
the  case  in  old  queens  Avhich  continue  to  lay  eggs  when  their 
receptaculum  contains  no  more  sperm;  and  workers,  which 
cannot  copulate  owing  to  the  rudimentary  development  of 
their  sexual  organs,  occasionally  lay  eggs  from  which  without 
exception  males  are  hatched. 

Dzierzon's  views  and  observations  were  confirmed  and 
completed  by  the  investigations  of  von  Siebold,  Leuckart,  and 
von  Berlepsch.^ 

Among  silkworm  breeders  the  opinion  had  been  repeatedly 
mooted  that  Bomhyx  mori  could  also  develop  from  the  unfertilized 
egg,  and  the  observations  made  on  this  point  by  von  Siebold 

*  According  to  the  recent  researches  of  E.  Bresslau,  the  case  is  still  more 
complicated. 

2  The  assertions  of  the  incorrectness  of  Dzierzon's  conclusions  which  have 
recently  been  vociferouslj^  maintained  have  been  proved  erroneous. 


Natural  Parthenogenesis  45 

and  by  others  in  addition  have  led  to  remarkable  results. 
Herold  had  already  observed  in  1838  that  a  certain  percentage 
of  the  unfertilized  eggs  of  the  silkworm  begin  to  develop,  but 
that,  in  contradistinction  to  the  fertilized  eggs,  the  development 
of  the  unfertilized  ones  comes  to  a  halt  in  the  first  stages,  and 
that  such  parthenogenetic  eggs  never  succeed  in  forming  cater- 
pillars.^ Schmid  and  von  Siebold-  observed  the  hatching  of 
caterpillars  from  unfertilized  eggs  of  Bombyx  mori,  and  these 
caterpillars  developed  into  sexually  mature  animals.  But 
the  results  of  other  observers  remained  in  part  contradictory. 
All  found  that  the  first  developmental  stages  occurred  also 
in  the  unfertilized  eggs,  but  in  the  majority  of  cases  the  eggs  died 
during  the  winter.  In  the  year  1871  von  Siebold  returned'^ 
to  the  question  of  parthenogenesis  in  Bombyx  mori  once  more, 
and  mentioned  the  investigations  of  Barthelemy.  This  author 
found  that  development  starts  very  much  later  in  the  unferti- 
lized eggs  of  Bombyx  mori  than  in  the  fertilized  ones. 

The  number  of  the  unfertilized  eggs,  in  which  the  actual  hatching 
of  the  caterpillar  through  parthenogenetic  development  was  reached, 
was  also  extraordinarily  variable;  only  once  in  Barthelemy's  investi- 
gations did  it  happen  that  nearly  all  the  unfertilized  eggs  of  a  virgin 
silkworm  developed,  while  those  cases  in  which  all  the  unfertilized  eggs 
laid  by  Bomhyx  mori  remained  sterile  were  very  abundant.  For  in 
cases  where  development  does  take  place  among  the  eggs  laid  by  a 
female  silkworm,  only  three  or  four  eggs  at  most  accompHsh  the  last 
stage  of  development,  i.e.,  the  hatching  of  a  caterpillar;  the  rest  remain 
at  various  earlier  degrees  of  development,  and  drj^  up. 

Further,  Barthelemy  remarks  that  these  strains  bred  from 
virgin  silkworms  proved  just  as  strong  as  those  produced  under 
the  influence  of  the  male  silkworm;  moreover,  these  individuals 
sprung  from  virgin  silkworms  showed  perfectly'  normal  sexual 
instincts.  Of  the  highest  importance  is  Barthelemy's  observa- 
tion that  only  virgin  silkworms  from  the  sunnner  brood  produce 
a  parthenogenetic  brood,  and  that  in  the  same  year;   while,  on 

1  After  von  Siebold.  ^  Von  Siebold,  op.  cit. 

*  Von  Siebold,  Beitrage  zur  Parthenogenese  der  Arthropoden.  LeipziR.  1871.  p.  232. 


46      Artificial  Parthenogenesis  and  Fertilization 

the  other  hand,  no  offspring  was  hatched  from  parthenogenetic 
eggs  of  either  the  summer  or  autumn  brood  when  kept  over 
winter.  Von  Siebold  brings  forward  observations  by  himself 
and  Schmid  which  confirm  Barthelemy's  latter  contention,  at 
least  in  part. 

These  investigations  on  silkworms  are  of  especial  interest 
to  us,  because  we  are  here  concerned  with  a  transition  form  in 
the  sense  that  the  eggs  of  the  silkworm  show  a  tendency  toward 
spontaneous  parthenogenesis;  but  it  depends  upon  certain  as 
yet  unknown  conditions  whether  or  not  the  eggs  develop  spon- 
taneously, i.e.,  without  fertilization,  and  whether  development 
stops  in  the  initial  stages,  or  whether  the  complete  development 
to  caterpillar  or  imago  takes  place.  This  is  supported  by  the 
fact  that  different  authors  have  achieved  such  contradictory 
results.  It  is  possible  that  not  only  external  circumstances, 
but  also  conditions  which  belong  to  the  egg  itself  (e.g.,  the 
nature  of  the  shell)  here  come  into  play.  We  shall  meet  later 
a  similar  case  in  the  parthenogenesis  of  starfish  eggs. 

Von  Siebold,  Leuckart,  and  other  authors  have  extended  the 
observations  on  natural  parthenogenesis  with  reference  to  the 
importance  which  these  observations  possess  for  another  funda- 
mental problem  of  biology — the  determination  of  sex.  In  the 
Psychidae  and  in  Solenohia  the  parthenogenetic  eggs  give  rise 
only  to  females,  in  bees  only  to  males.  Leuckart  and  von 
Siebold  discovered  that  in  other  hymenoptera  too  (Polistes, 
Vespttf  and  Nematus)  similar  things  take  place,  and  that  here 
too  parthenogenesis  occurs,  leading,  however,  exclusively  to 
the  production  of  males.  In  Crustacea,  such  as  Apus,  Artemia, 
and  Luimadia,  von  Siebold  likewise  found  parthenogenetic 
development;  but  here  it  led  to  the  production  of  fenlales  only. 

This  sketch  of  the  earlier  observations  upon  spontaneous 
parthenogenesis  will  suffice  for  the  comprehension  of  the  ex- 
periments on  artificial  parthenogenesis  with  which  they  are 
historically  connected. 


VI 

ON  THE  HISTORY  OF  THE  EARLIER  EXPERIMENTS  ON 
ARTIFICIAL  PARTHENOGENESIS 

The  observations  upon  the  natural  parthenogenesis  of 
Bomhyx  7non  were  the  starting-point  for  investigations  upon 
artificial  parthenogenesis.  In  the  year  1847  a  French  author, 
Boursier,  stated  ''that  he  had  placed  a  female  silkworm  moth, 
which  had  not  paired  with  a  male,  first  in  the  sunlight  and  then 
in  the  shade,  where  (in  both  cases)  it  had  laid  many  eggs. 
Caterpillars  had  been  produced  from  each  of  those  eggs  laid 
in  the  sunlight."^ 

Von  Siebold  comments  upon  this:  ''Since  in  the  foregoing 
cases  nobody  attributed  the  fertilization  of  the  eggs  to  the 
influence  of  the  light  and  warmth  of  the  sun,  as  Boursier  has 
done,  one  cannot  abstain  from  regarding  this  phenomenon  as  a 
case  of  parthenogenesis."  Considering  the  above-mentioned 
observation  of  Barthelemy  upon  the  difference  in  behavior  of 
the  summer  and  winter  eggs,  and  also  the  theoretical  results 
of  the  investigations  upon  artificial  parthenogenesis  to  be 
mentioned  later,  it  is  a  priori  not  impossible  that  the  tempera- 
ture to  which  the  newly  laid  egg  of  Bomhyx  mori  is  exposed 
may  be  of  importance  for  its  development. 

In  the  year  1886,  Tichomiroff-  published  a  short  note  on 
"Artificial  Parthenogenesis  in  Insects,"  the  object  of  which  was 
to  lend  fresh  support  to  the  work  of  Herold  and  von  Siebold 
upon  natural  parthenogenesis  in  the  silkworm.  "It  has  long- 
been  known  that  the  eggs  of  Bomhyx  mori  can  develop  partheno- 
genetically;  yet  one  is  always  hearing  doubts  expressed  about 

I  Von  Siebold,   Wahre  Parthenogenese,  p.  126,  Leipzig,  1856. 

-A.  Tichomiroff,  "Die  IcUnstliche  Partlienogenese  bei  Iiisekten,"  Arch.  f. 
Anat.  u.  Physiol.,  Physiol.  Abt.,  1886,  Suppl.,  p.  35;  Zoologischer  Anzeiger,  XXV, 
386,  1902. 

47 


48      Artificial  Parthenogenesis  and  Fertilization 

it,  even  after  the  work  of  Herold  and  von  Siebold."  In  order 
to  increase  the  number  of  Bomhyx  eggs  that  develop  without 
fertihzation,  Tichomiroff  used  a  method  of  which  the  breeders 
avail  themselves  in  order  to  accelerate  the  development  of 
fertilized  eggs.  Certain  kinds  of  silkworms  lay  their  eggs  in 
the  summer  and  these  eggs  begin  to  develop  at  once;  but 
during  the  winter  the  development  ceases,  and  the  caterpillars 
do  not  hatch  till  the  spring.  Now  it  appears  that  it  is  cus- 
tomary among  the  breeders  to  hasten  the  development  of  the 
fertilized  eggs  by  special  "stimuh,"  so  that  the  caterpillars 
hatch  out  in  the  same  summer  in  which  the  eggs  are  laid. 
Tichomiroff  applied  the  same  methods  to  unfertilized  eggs. 

The  experiments  consisted  in  stimulating  eggs  mechanically  and 
chemically  in  the  same  way  as  is  done  in  order  to  obtain  caterpillars 
in  the  same  summer  from  fertilized  eggs  which  normally  only  develop 
to  a  certain  stage  in  summer.  I  plunged  thirtj^-six  unfertiHzed  eggs 
into  concentrated  sulphuric  acid  and  left  them  there  two  minutes 
(afterward  the  eggs  were  scrupulously  washed).  Thirteen  of  these 
eggs  began  to  change  color  on  the  fourteenth  da3\  On  the  sixteenth 
day  an  embryo  could  be  perceived  in  these  eggs.  Both  the  embrj^o  and 
the  serous  envelope  consisting  of  magnificent  pigment  cells  appeared 
quite  normal. 

Sixteen  other  eggs  were  rubbed  quite  lightly  with  a  brush.  Up  till 
now  (after  one  week  [?])  the  result  has  remained  negative:  not  a 
single  egg  has  developed.  A  third  batch  of  ninety-nine  eggs  were 
brushed  hard.  On  the  fourth  day  the  color  change  characteristic 
of  developing  eggs  was  observed  in  six  of  these  eggs.  Xot  a  single 
parthenogenetically  developing  egg  was  observed  among  all  the 
unfertihzed  eggs  which  remained  unstimulated. 

The  breeders'  experience  that  the  immersion  of  eggs  in 
concentrated  sulphuric  acid,  or  brushing  them,  accelerates 
development,  is  hard  to  explain.  Both  rubbing  the  eggs  with 
a  brush  and  plunging  them  into  sulphuric  acid  may  serve, 
perhaps,  to  injure  or  alter  the  skin  of  the  egg,  whereby  it 
becomes  more  permeable  to  oxygen.     We  shall  see  later  that 


History  of  Artificial  Parthenogenesis  49 

the  egg  of  the  frog  can  be  induced  to  develop  parthenogeneti- 
cally  by  a  puncture  with  a  needle.  It  is  not  impossible  that  in 
the  experiments  mentioned  here  on  silkworms  also  a  rupture 
or  puncture  of  the  surface  layer  was  the  cause  of  the  develop- 
ment. 

Tichomiroff's  researches  caused  Dewitz^  to  interpret  as 
parthenogenesis  a  previous  observation  of  his  upon  frog  eggs. 

When  I  was  working  in  the  spring  of  1885  under  Professor  Zuntz 
in  the  physiological  institute  of  the  agricultural  college  in  Berlin,  I 
placed,  for  certain  reasons,  unfertilized  eggs  of  Ranafusca  in  a  solution 
of  corrosive  sublimate.  The  next  morning,  to  my  astonishment, 
I  found  them  swollen  and  segmented.  In  some  of  the  eggs  only  one 
division  had  taken  place,  in  others  more  than  one ;  in  some  the  cleavage 

was  irregular,  but  in  very  many  perfectly  normal Moreover, 

this  occurred  just  as  well  if  the  eggs  remained  lying  in  the  sublimate 

as  also  when  they  had  remained  there'  only  a  few  minutes 

Hence  it  can  be  concluded  that  subhmate  exerts  a  stimulus  which  causes 
the  first  division. 

There  can  be  no  doubt  that  the  last  conclusion  is  incorrect. 
What  Dewitz  observed  was  obviously  a  coagulation  phenome- 
non which  led  to  a  wrinkling  of  the  surface  of  the  egg  and  in 
which  the  essence  of  cell  division,  viz.,  nuclear  division,  was 
absent.     This  was  afterward  pointed  out  by  Rolix.^ 

In  the  eighties  and  nineties,  the  attention  of  morphologists 
was  directed  to  the  finer  processes  of  nuclear  and  cell  division. 
This  line  of  work  yielded  to  the  domain  of  facts  in  which  we 
are  interested  here,  the  observation  that  the  unfertihzed  eggs  of 
certain  sea-urchins  may  show  the  beginning  of  nuclear  or  even 
a  cell  division  if  they  remain  long  enough  in  sea-water.  The 
first  systematic  observation  on  this  count  probably  originated 
from  Richard  Hertwdg.^ 

1  J.  Dewitz,  "Kurze  Notiz  ueber  die  Furchung  von  Froscheiern  in  Sublimat- 
losung,"  Biol.  Centralbl.,  VII,  93,  1888. 

2  W.  Roux,  Gesammelte  Ahhandl.,  II,  432. 

3  R.  Hertwig,  "Ueber  die  Entwicklung  des  unbefruchteten  Seeigeleies," 
Festschrift  fiir  Gegenbaur,  Leipzig,  1896.  (Hertwig  had  already  reported  on  these 
observations  in  the  German  Zoological  Association  in  1892.) 


50      Artificial  Parthenogenesis  and  Fertilization 

When  in  the  spring  of  1887  my  brother  and  I  performed  experi- 
ments upon  the  fertihzation  of  sea-urchin  eggs,  we  had  before  us  the 
question  as  to  what  influence  the  concentration  of  sperm  exerted  upon 
the  polyspermous  fertilization  of  injured  eggs,  and  especially  those 
whose  life  functions  had  been  interfered  with,  through  treatment  with 
reagents.  Thus  eggs  were  treated  for  thirty  minutes  with  a  1  per  cent 
solution  of  strj^chnin  and  then  mixed  with  sperm  of  the  same  species 
at  various  dilutions.  In  one  case  the  sperm  was  so  much  diluted  with 
sea-water  that  on  the  evidence  both  of  observation  in  the  living  con- 
dition and  of  a  very  thorough  examination  of  preserved  material  more 
than  90  per  cent  remained  unfertihzed  owing  to  the  addition  of  insuffi- 
cient sperm.  Fifty  minutes  after  fertilization  the  control  material 
was  preserved.  As  before,  89  per  cent  of  them  were  unfertihzed.  In 
these  I  found  the  beginning  of  an  interesting  change,  which  I  will 
here  describe. 

Briefly,  this  alteration  consisted  in  the  fact  that  the  nucleus 
show'cd  changes  similar  to  those  occurring  after  fertilization. 
The  division  processes  started  in  the  nucleus,  but,  ''in  the 
majority  of  cases  a  nuclear  or  cell  division  did  not  take  place. 
In  exceptional  cases  a  single  division  of  the  egg  into  two  cells 
occurred,  and  each  of  these  was  provided  with  a  nucleus.  These 
cases  which  approach  nearest  to  a  normal  division  process  are 
rare,  and  they  also  differ  from  the  normal"  (p.  44).  Later 
Hertwig  convinced  himself  that  the  longer  sea-urchin  eggs  had 
lain  in  sea-water  without  the  addition  of  sperm,  the  more  of 
such  divisions  incidentally  occurred.  We  shall  later  return  to 
this  phenomenon. 

In  1892  the  writer^  found  that  if  the  freshly  fertilized  eggs 
of  a  sea-urchin  (Arbada)  are  brought  into  hypertonic  sea-water 
(about  100  c.c.  of  sea-water +2  g.  NaCl)  the  eggs  do  not  divide 
in  such  a  solution;  but  if  they  are  returned  to  normal  sea-water 
after  tw^o,  three,  or  four  hours,  in  quite  a  short  time,  after  twenty 
or  even  ten  minutes,  the  egg  breaks  up  into  several  cells  at 
once;  and  indeed  the  longer  the  eggs  remain  in  the  hyper- 
tonic solution,  the  greater  the  number  of  the  cells  into  which 

1  Loeb,  "Experiments  on  Cleavage,"  Joiir.  Morphol.,  VII,  253,  1892. 


History  of  Artificial  Parthenogenesis  51 


they  suddenly  divide.  Since  cell  division  follows  nuclear  divi- 
sion, I  concluded  that  in  this  experiment  nuclear  division  went 
on  in  the  hypertonic  solution,  while  cell  division  was  stopped. 
I  held  the  loss  of  water  which  the  egg  had  suffered  in  the 
hypertonic  solution  answerable  for  the  prevention  of  cell 
division.  If  the  eggs  remain  too  long  in  the  hypertonic  solution 
or  if  its  concentration  is  too  high,  nuclear  division  also  comes 
to  a  stop.  W.  W.  Norman^  later  undertook  a  histological 
examination  of  such  eggs  and  confirmed  this  conclusion. 

I  suggested  that  the  hj^pertonic  solution  produces  a  kind 
of  ''rigor"  in  the  cytoplasm  by  the  withdrawal  of  water,  and 
that  in  consequence  the  movements  necessary  for  cell  division 
can  no  longer  proceed.  If  one  adds  a  little  more  NaCl  to  the 
sea-water  than  is  absolutely  necessary  to  stop  cell  division, 
the  breaking-up  of  the  nucleus  does  not  take  place  either,  for 
movements  of  the  protoplasm  are  very  probably  necessary  for 
this  also.  This  explains  the  results  of  Morgan,^  who  repeated 
my  experiments,  but  observed  no  nuclear  division.  He  found, 
however,  as  I  did,  that  eggs  treated  for  the  same  time  with 
hypertonic  sea-water  suddenly  divide  into  several  cells  about 
ten  minutes  after  they  have  been  returned  to  normal  sea-water. 
Obviously  he  was  deahng  with  eggs  in  which  the  changes  in  the 
nucleus  necessary  for  segmentation  had  all  taken  place,  but  in 
which  the  movements  of  the  protoplasm  necessary  for  the  pulling 
apart  of  the  chromosomes  had  been  suppressed.  Morgan 
observed,  however,  that  the  astrospheres  were  formed  in  these 

eggs.^ 

At  the  same  time  Mead  also,  independently  of  Morgan,  and 
from  another  point  of  view,  attacked  experimentally  the 
question  of  the  importance  of  the  centrosomes.  Boveri  had 
put  forward  the  view  that  the  unfertilized  egg  is  unable  to 

1  Norman,    Archiv  f.   Entwicklungsmechanik,  III,   106,    1896. 

2  Morgan,    Anat.   Anzeiger,  IX,   149,   1894. 

3  Morgan,  "The  Production  of  Artificial  Astrospheres."  Archiv  f.  Entwicklungs- 
mechanik, III,  339,  1896. 


52       Artificial  Parthenogenesis  and  Fertilization 

develop  because  it  lacks  the  apparatus  for  cell  division — the 
centrosome.  According  to  Boveri,  the  spermatozoon  starts 
the  development  of  the  egg  by  introducing  into  it  a  centrosome. 
The  egg  is  thus  placed  in  a  position  to  begin  its  development. 
Only  the  centrosome  can  be  the  cause  of  division.  Now  Mead 
points  out  that  in  certain  eggs,  e.g.,  that  of  Chaetopterus, 
the  spermatozoon  has  quite  a  different  effect.  As  long  as  the 
egg  of  Chaetopterus  is  in  the  ovary,  no  polar  bodies  are  given 
off;  but  as  soon  as  it  reaches  sea-water  maturation  starts  and 
a  spindle  is  formed  preliminary  to  the  extrusion  of  the  first 
polar  body. 

The  egg  remains  at  this  stage  so  long  as  no  spermatozoon 
enters  it. 

In  the  egg  of  Chaetopterus  a  perfect  amphiaster  with  centrosomes, 
centrospheres,  astral  rays,  and  spindle  fibres  is  developed  and  the 
egg  remains  for  hours  in  the  metaphase,  if  it  is  left  unfertilized  in  sea- 
water;  and  the  same  appears  to  be  true  of  many  other  marine  annelids. 
This  elaborate  machinery  of  mitotic  division  is  immediately  set  in 
motion  upon  the  entrance  of  the  spermatozoon,  though  the  sperm  and 
its  centrosomes  are  in  a  distant  portion  of  the  egg.  All  the  phases  of 
this  and  the  subsequent  mitosis  are  independent  of  the  karyokinetic 
changes  in  the  vicinity  of  the  sperm.  Since  in  one  form  the  oocyte 
will  not  divide  until  the  sperm  enters  the  cell,  even  though  the  centro- 
somes and  the  whole  amphiaster  are  present,  the  suspicion  is  warranted 
that  in  the  ripe  egg  of  other  forms — the  sea-urchin,  for  example — the 
mitosis  is  not  inhibited  merely  on  account  of  the  lack  of  a  centrosome, 
nor  is  it  incited  merely  because  a  new  centrosome  is  introduced  to 
organize  the  mitotic  figure.^ 

Mead  concludes  that  for  cell  division  "a  stimulus  is  required, 
analogous,  perhaps,  to  that  which  starts  into  activity  the 
motor  apparatus  of  pigment  cells,  leucocytes  or  muscle  cells." 
In  order  to  pursue  this  idea  farther  he  made  some  experiments 
upon  the  effect  of  salts,  on  the  advice  of  C.  W.  Green.  He 
emploj^ed  the  salts  of  the  Ringer  solution  and  found  that  if 

» A.  D.  Mead,  "The  Rate  of  Cell  Division  and  the  Function  of  the  Centro- 
some," Biological  Lectures  delivered  at  Woods  Hole,  1896-97,  p.  211,  Boston,  1898. 


History  of  Artificial  Parthenogenesis  53 

a  small  amount  (J  to  J  of  1  per  cent)  of  KCl  is  added  to  the 
sea-water,  the  amphiaster  of  the  first  oocyte  stage  at  once 
resumes  its  activity.  ''The  maturation  processes,  including 
the  extrusion  of  the  first  and  second  polar  bodies  and  the 
concomitant  changes  in  the  form  of  the  egg,  succeed  one  another 
with  the  same  regularity  that  obtains  when  the  egg  is  fertilized." 
Mead,  however,  observed  no  further  development  in  these 
eggs.  From  these  experiments  he  concludes  that,  in  the  normal 
fertilization  of  Chaetopterus,  "the  entering  sperm  stimulates 
these  mitotic  activities  in  a  similar  manner,  i.e.,  by  exerting 
a  chemical  influence  on  the  egg  and  not  by  furnishing  the  egg 
with  the  organs  of  division."  This  work  of  Mead's  which,  in 
my  opinion,  occupies  a  distinguished  position  among  cytological 
treatises,  has  been  but  little  noticed  in  the  literature  of  the 
subject. 

In  1899  Morgan^  published  some  new  and  important  obser- 
vations on  the  effect  of  hypertonic  solutions  upon  the  unferti- 
lized egg.  He  had  discovered  that  if  unfertilized  eggs  were 
treated  with  hypertonic  sea-water,  they  began  to  divide  ^vithout, 
however,  developing  into  larvae.  Morgan  considered  this 
cleavage  an  abnormal  phenomenon,  which  was  in  no  way  com- 
parable to  normal  segmentation.  ''The  form  of  the  cleavage 
is  totally  different  from  that  of  the  normal  cleavage."  Accord- 
ing to  Morgan's  illustrations,  the  eggs  appear  to  have  developed 
to  about  the  sixteen-cell  stage,  but  not  farther.  "The  time 
that  it  takes  for  a  cleavage  plane  to  pass  through  the  egg  is 
often  very  long  in  comparison  to  the  time  of  normal  division. 
The  result  is  a  mass  of  extremely  minute  granules  or  pieces. 
These  pieces  never  acquire  cilia  and  do  not  produce  any  form 
that  resembles  any  stage  of  the  normal  embryo.  Later  the 
masses  disintegrate"  (pp.  454  and  455).  Morgan's  aim  was  not 
to  obtain  artificial  parthenogenesis,  but  to  corroborate  his  former 

1  Morgan,  "The  Action  of  Salt  Solutions  on  the  Unfertilized  and  Fertilized 
Eggs  of  Arbacia  and  of  Other  Animals,"  Archiv  f.  E ntwicklungsmechanik,  VIII.  448 
1899. 


54      Artificial  Parthenogenesis  and  Fertilization 

statement  that  the  effect  of  the  hypertonic  solutions  upon  the 
egg  consisted  in  the  production  of  artificial  astrospheres  and 
centrosomes,  and  that  such  astrospheres  and  centrosomes  were 
the  organs  of  cell  division.  He  considered  that  a  development 
of  unfertilized  eggs  treated  with  hypertonic  sea-water  to  larvae 
was  quite  excluded. 

Meanwhile  I  had  been  led  by  the  results  of  my  investiga- 
tions upon  the  effects  of  ions  to  the  question  whether  it  might 
not  be  possible  to  cause  unfertilized  eggs  to  develop  into  larvae 
by  treating  them  with  modified  sea-water.  Experiments  upon 
the  physiological  effect  of  the  galvanic  current  had  led  me  to 
the  conclusion  that  in  such  cases  we  are  dealing  with  an  ion 
effect — an  idea  which  was  new  at  that  time  but  which  is  con- 
sidered a  matter  of  fact  today — and  since  the  galvanic  current 
is  a  sovereign  method  of  stimulating  muscle  and  nerve,  it  gave 
me  the  idea  that  perhaps  ion  effects  might  really  underlie  the 
action  of  all  stimuli  which  had  not  hitherto  been  closely  ana- 
lyzed. Ringer's  and  my  own  experiments  with  certain  salts 
showed  that  solutions  of  sodium,  lithium,  calcium,  and  rubidium 
salts  could  send  skeletal  muscles  into  rhythmic  or  at  least 
fibrillar  contractions,  while  salts  of  calcium,  magnesium,  and 
strontium  prevented  these  contractions.  Hence  we  owe  it 
to  the  presence  of  the  calcium  salts  or  ions  in  our  blood  that 
our  muscles  do  not  continually  contract. 

Now  as  mentioned  above,  it  had  been  made  known  by  Hert- 
wig  and  others,  and  I  myself  had  often  enough  observed  in 
Arbacia,  that  the  unfertilized  eggs  of  certain  animals  occasion- 
ally begin  to  divide  provided  they  remain  long  enough  in  sea- 
water.  This  suggested  that  the  unfertilized  egg  might  be  in 
just  the  same  condition  as  the  muscle  in  that  the  egg  could 
develop  without  fertilization,  but  that  something  contained 
in  the  sea-water  prevents  its  development,  just  as  the  Ca 
and  Mg  contained  in  the  blood  prevent  fibrillar  contractions 
of  the  muscles.     Hence  I  decided  to  perform  experiments  in 


History  of  Artificial  Parthenogenesis  55 

order  to  see  whether  it  were  not  possible  to  cause  unfertiHzed 
sea-urchin  eggs  to  develop  into  larvae  by  altering  the  constitu- 
tion of  the  sea-water.  Mv  chief  idea  was  that  it  must  take 
place  by  means  of  hydroxylions  or  of  hydrions.  For  earlier 
experiments  had  shown  that  an  addition  of  acid  to  sea-water 
retards  the  development  of  sea-urchin  eggs.^ 

Moreover,  it  was  conceivable  that  the  alkali  of  the  sea- 
water  or  the  formation  of  carbonic  acid  in  the  unfertilized  eggs 
was  itself  the  cause  that  they  began  occasionally  to  divide 
spontaneously  after  a  prolonged  sojourn  in  sea-water.  So  I 
then  tried  whether  it  was  possible  to  cause  the  development 
of  the  unfertilized  sea-urchin  egg  into  a  larva  by  means  of  alka- 
lies or  acids.^  My  later  investigations  have  shown  that  I 
was  right  in  this  idea,  but  through  a  curious  accessory  circum- 
stance I  did  not  at  that  time  succeed  in  my  object.  For  I 
was  then  of  the  opinion  that  onty  the  hydrions  are  of  impor- 
tance in  the  physiological  effect  of  acid,  and  hence  I  used  only 
strong  mineral  acids.  I  have  since  found  that  in  this  case  only 
the  weak  acids  are  effective.  Had  I  used  fatty  instead  of 
mineral  acids,  the  development  of  this  branch  of  biolog>'  would 
have  been  shortened  by  five  years. 

On  the  other  hand  the  experiments  on  the  addition  of  ]MgCl_. 
to  the  sea-water  were  successful,  provided  that  at  the  same 
time  the  osmotic  pressure  of  the  sea-water  was  raised.^  Unferti- 
lized sea-urchin  eggs  which  had  been  exposed  for  two  hours  to 
such  a  mixture  developed  afterward  into  larvae,  some  of  which 
reached  the  normal  pluteus  stage.  In  this  way  the  first  step 
was  taken  which  rendered  possible  the  systematic  analysis  of 
the  nature  of  the  process  of  fertilization.     For  we  possess  only 

1  Loeb,  "  Uel:)er  den  Einfluss  von  Sauren  und  Alkalien  auf  die  embryonale 
Eiitwicklung  und  das  Waclistum,"  Archiv  /.  Entwicklungsmechanik,  VII,  631,  189S. 

-Loeb,  Am.  Jour.  Physiol.,  III.  434,  1900. 

3  Loeb,  "On  the  Nature  of  the  Process  of  Fertilization,  and  the  Production 
of  Normal  Larvae  (Plutci)  from  the  Unfertilized  Eggs  of  the  Sea-Urchin,"  .Iw. 
Jour.   Physiol.,  Ill,    135,    1S99. 


56      Artificial  Parphexogenesis  and  Fertilization 


a  single  criterion  which  allows  us  to  decide  whether  an  agent 
has  an  effect  upon  the  egg  similar  to  that  of  the  entry  of  the 
spermatozoon;  this  criterion  is  the  development  of  the  egg 
into  a  larva.  The  mere  initiation  of  cell  division  is  not  sufficient 
since,  for  example,  cell  divisions  occur  in  the  cases  of  the  growth 
of  tumors  and  galls,  which  do  not  lead  to  the  formation  of  a 
larva.  This  distinction  between  a  cell  division  which  forms  the 
basis  of  normal  development  and  growth  and  one  which  leads 
to  the  formation  of  pathological  products  is  also  of  practical 
importance. 

It  is  not  our  purpose  in  this  book  to  report  all  the  investi- 
gations upon  artificial  parthenogenesis.  We  shall  rather  con- 
fine ourselves  to  those  experiments  which  help  us  to  obtain  an 
insight  into  the  physicochemical  character  of  the  process  of 
development.  We  will  start  with  the  experiments  upon  sea- 
urchin  eggs,  which  appear  to  be  best  suited  to  such  investiga- 
tions. The  forms  of  sea-urchins  with  whose  eggs  I  have  worked 
are  Arhacia  of  the  Atlantic  Ocean  (at  Woods  Hole)  and  Strongy- 
locentrotus  purpuratus  and  franciscanus  of  the  Pacific  Ocean  (at 
Pacific  Grove). 

The  eggs  of  both  these  forms  develop  only  when  incited 
thereto  either  by  sperm  or  by  the  chemical  methods  hereafter 
to  be  described. 


VII 

THE  FIRST  EXPERIMENTS  UPON  THE  OSMOTIC  ACTIVA- 
TION OF  THE  UNFERTILIZED  EGG  OF  THE 
SEA-URCHIN  (Arbacia) 

1.  As  already  mentioned,  I  began  my  investigations  with 
the  anticipation  that  it  must  be  possible  to  induce  the  un- 
fertilized eggs  to  develop  by  treating  them  with  bases  or  acids. 
For  several  weeks  in  the  summer  of  1899  I  conducted  experi- 
ments in  this  direction  on  the  eggs  of  a  sea-urchin,  Arbacia, 
at  Woods  Hole,  without  obtaining  any  other  result  than  that 
the  unfertihzed  eggs  of  Arbacia  placed  in  100  c.c.  of  sea-water + 

1  c.c.  of  N/10  NaOH  begin  to  segment  after  remaining  in  the 
solution  for  about  five  hours.  The  cleavage,  however,  was  very 
irregular  and  did  not  go  beyond  the  early  stages — two  or  four 
cells.  At  the  same  time  the  eggs  showed  a  tendency  to  become 
amoeboid.  The  experiments  with  acids  (HCl,  HNO3,  H2SO4) 
showed  that  no  cleavage  took  place  in  acidified  sea-water,  but 
that  a  few  divisions  might  be  observed  in  unfertilized  eggs  if 
they  were  placed  for  about  ten  minutes  in  100  c.c.  of  sea-water + 

2  or  3  c.c.  of  N/10  HCl  and  then  replaced  in  normal  sea-water.^ 
Experiments  with  salt  solutions  which  were  isosmotic  with 
sea-water  gave  no  better  results.  When  the  summer  of  1899 
had  almost  entirely  elapsed  in  this  manner  without  any 
success,  I  at  last  investigated  the  effects  of  hypertonic  solutions. 
10/8  m  (grammolecular)  solutions  of  NaCl,  KCl,  CaCla,  and 
MgCls,  were  prepared  and  mixed  in  different  proportiorus 
with  sea-water.  After  prolonged  experiments  I  found  that  if 
unfertilized  sea-urchin  eggs  were  exposed  for  two  hours  to  a 
mixture  of  50  c.c.  of  sea-water+50  c.c.  10/8  m  MgCl.>  and  then 

1  Loeb,  "On  the  Artiflcial  Production  of  Normal  Larvae  from  the  UnfortiUzod 
Eggs  of  the  Sea-Urchin,"  Am.  Jour.  Physiol.  Ill,  434,  1900;  Untersuchungen 
zur  kiinstlichen  Parthenogenese,  p.  77,   1906. 

57 


58      Artificial  Parthenogenesis  and  Fertilization 

replaced  in  normal  sea-water  they  developed  into  swimming 
plutei.^  I  expected  that  the  same  result  would  also  be  obtained 
with  NaCl,  KCl,  and  CaCl2,  but  to  my  surprise  this  was  not  the 
case.  Meanwhile  since  the  spawning  season  had  expired,  and 
there  were  less  than  a  dozen  sea-urchins  at  my  disposal,  I  used 
this  material  to  confirm  at  least  the  main  and  most  important 
result,  that  it  is  really  possible  to  produce  larvae  from  unferti- 
lized sea-urchin  eggs.  Eight  series  of  experiments,  each  with  a 
large  number  of  different  solutions,  which  I  was  still  able  to 
carry  out  together  with  many  control  experiments,  convinced 
me  that  I  had  succeeded  in  the  artificial  production  of  larvae 
from  unfertilized  eggs.  Not  only  blastulae  but  also  gastrulae 
and  plutei,  some  of  them  entirely  normal  in  appearance,  had  been 
produced.  Then,  however,  the  further  question  arose  whether 
this  was  an  effect  of  the  hj^pertonic  sea-water  alone  or  whether 
a  specific  action  of  magnesium  was  responsible  for  the  result. 
Lack  of  material  made  it  impossible  for  me  to  decide  this 
question  the  same  summer  at  Woods  Hole.  In  February,  1900, 
I  took  up  this  investigation  at  Pacific  Grove  on  the  California 
coast.  Dr.  Garrey,  my  assistant  at  that  time,  accompanied 
me;  and  we  were  able  to  prove  that  an  increase  of  concentration 
of  sea-water,  not  only  by  MgCl2,  but  also  by  NaCl  and  sugar, 
incited  the  development  of  the  sea-urchins  of  that  coast — 
Strongylocentrotus  purpuratus  and  franciscaniis.  We  made  sure 
also  of  the  fact  that  the  most  scrupulous  sterilization  of  the  sea- 
water  and  of  the  instruments  and  the  elimination  of  all  possible 
sources  of  error  did  not  invalidate  the  results.  However,  besides 
these  gratifying  results  we  also  had  a  very  unwelcome  experience 
which  long  remained  inexplicable  to  me :  The  results  in  Pacific 
Grove  were  not  so  constant  as  in  Woods  Hole.  On  some  days 
the  experiments  went  very  well,  but  then  there  followed  days  on 
which  the  same  solution  which  had  hitherto  given  good  results 

» In  the  previously  quoted  detailed  account  of  this  first  investigation  (1900) 
I  expressly  mentioned  that  the  raising  of  the  osmotic  pressure  of  the  solution  was 
a  necessary  condition  of  the  experiment. 


Osmotic  Parthenogenesis  59 

remained  entirely,  or  almost  entirely,  ineffectual.  I  could  at 
that  time  assign  no  reason  for  this,  and  postponed  the  publica- 
tion of  these  results  until  T  had  the  opportunity  of  repeating 
them  once  more  in  Woods  Hole.  In  the  summer  of  1900  I 
convinced  myself  at  Woods  Hole  that  for  Arbacia  also  Mg  ions 
play  no  specific  role,  but  that  it  is  merely  a  case  of  appropriate 
increase  of  osmotic  pressure.^  As  long  as  the  osmotic  pressure 
of  sea-water  is  raised  about  50  per  cent,  it  is  immaterial  whether 
the  rise  of  pressure  is  caused  by  electrolytes  like  MgCl2,  NaCl, 
KCl,  or  CaCl2,  or  by  the  addition  of  non-electrolytes  such  as 
cane  sugar  and  urea.  The  experiments  upon  the  eggs  of  Arbacia 
at  Woods  Hole  gave  much  more  constant  results  than  the 
experiments  on  Strongylocentrotus  in  Pacific  Grove.  The ." 
reason  that  in  the  previous  year  artificial  parthenogenesis  was  , 
successful  only  when  the  concentration  of  the  sea-water  was 
raised  by  MgCls  was  due  to  the  fact  that  the  solutions  of 
salts  with  which  I  worked  were  not  isosmotic,  as  I  had  sup- 
posed. 

Now  as  in  this  treatise  we  are  especially  interested  in  the 
quantitative  aspect  of  this  experiment,  we  must  discuss  some- 
what more  thoroughly  the  amount  of  increase  of  osmotic 
pressure  that  is  necessary  for  development.  According  to 
W.  E.  Garrey,  in  the  sea-water  at  Woods  Hole  the  freezing- 
point  is  lowered  by  1.818°,  while  the  water  flowing  in  the 
laboratory  has  a  somewhat  higher  concentration,  viz.,  A  = 
1.83°.^  Freezing-point  determinations  on  pure  NaCl  solutions 
gave  for  a  m/2  NaCl  solution  A  =  1 .  75  °,  and  for  a  m/2  van't 
Hoff  solution  (i.e.,  for  a  mixture  of  100  c.c.  m/2  NaCl: 2.2  c.c. 
m/2  KCl: 2  c.c.  m/2  CaCl2:12c.c.  m/2  MgCU)  the  lowering 
of  the  freezing-point  is  somewhat  greater,  viz.,  about  1.84°. 

1  Loeb,  "Further  Experiments  on  Artificial  Parthenogenesis  and  the  Nature 
of  the  Process  of  Fertilization,"  Am.  Jour.  Physiol.  IV,  178,  1900;  Untersuchungen 
ueber  kunstliche  Parthenogenese,  p.  154. 

2W.  E.  Garrey,  "The  Osmotic  Pressure  of  Sea- Water  and  of  the  Blood  of 
Marine  Animals,"   Biol.   Bull.,  VIII,  257,   1905. 


60      Artificial  Parthenogenesis  and  Fertilization 

This  is  due  to  the  fact  that  MgCl2  and  CaCl2  dissociate  into 
three  ions.  In  sea-water,  however,  there  are  not  12  c.c.  of 
MgClg,  but  7.8  c.c.  MgCl2+3.8c.c.  MgS04,  and  hence  a  really 
m/2  van't  Hoff  solution  has  a  little  weaker  osmotic  pressure. 
At  the  same  time,  the  difference  in  osmotic  pressure  between 
a  van't  Hoff  solution  and  a  m/2  NaCl  solution  is  small. ^ 

Now  I  found  that  in  Woods  Hole  artificial  parthenogenesis 
may  be  produced  in  sea-urchin  eggs  if  they  are  left  for  between 
one  and  two  hours  in  a  mixture  of  90  c.c.  of  sea-water +10  c.c. 
of  2 J  m  NaCl  or  KCl.  The  increase  of  osmotic  pressure  of  this 
solution  was  relatively  small,  amounting  to  about  40  per  cent 
of  the  osmotic  pressure  of  the  sea-water.^  I  also  obtained 
swimming  larvae  when  the  eggs  of  Arbacia  were  put  for  about 
two  hours  in  the  following  solutions: 

100  c.c.  of  sea-water+25  c.c.  2  m  cane  sugar. 
80  c.c.  of  sea-water +17|  c.c.  2^  m  urea. 

In  this  case  also  only  a  very  slight  increase  of  molecular  con- 
centration takes  place. 

A  pure  cane-sugar  solution  of  relatively  low  molecular  con- 
centration is  alone  sufficient  to  cause  unfertilized  eggs  of  sea- 
urchins  to  develop.  Thus  eggs  of  Arbacia  were  placed  for  two 
hours  in  a  solution  of  60  c.c.  2  m  cane  sugar +40  c.c.  distilled 
water  or  even  in  55  c.c.  2  m  cane  sugar+45  c.c.  distilled  water^ 
In  the  last  case  there  is  only  a  very  slight  increase  of  molecular 
concentration.^  Nevertheless  this  treatment  activates  the  egg, 
though  its  development  never  goes  farther  than  the  blastula 
stage,  and,  as  a  rule,  not  even  so  far.  The  development  of 
the  egg  generally  ceases  in  the  early  segmentation  stages.  We 
shall  see  later  that  the  production  of  plutei  is  favored  if  the 

1  According  to  Garrey,  the  sea-water  at  Pacific  Grove  lowers  the  freezing- 
point  by  1 .  90°. 

2  Am.  Jour.  Physiol.,  IV,  178,  1900.  ^  Jbid. 

4  We  shall  see  later  that  the  osmotic  effect  of  a  pure  cane-sugar  solution  is 
considerably  higher  than  its  theoretically  calculated  value. 


Osmotic  Parthenogenesis  01 

hypertonic  solution  possesses  a  rather  high  concentration  of  HO 
ions.  This  is  more  important  for  the  eggs  of  S.  purpuratus 
than  for  those  of  Arbacia,  since  the  latter  can  be  caused  to 
develop  even  by  a  neutral  hypertonic  solution.  We  pointed 
out  already  that  the  eggs  of  Arbacia  require  for  their  develop- 
ment a  lower  concentration  of  hydroxylions  than  the  eggs  of 
purpuratus.  I  published  the  sugar  experiment  in  order  to 
leave  no  doubt  that  the  hypertonic  solution  produces  its  effect 
only  in  virtue  of  its  capacity  for  withdrawing  water,  and  that 
we  are  not  dealing  with  a  specific  action  of  salts  or  their  ions.^ 

This  series  of  investigations  established  still  another  point  of 
theoretical  importance.  The  egg  loses  water  in  the  hyper- 
tonic solution;  but  when  replaced  into  normal  sea-water  it 
naturally  takes  up  water  again.  The  question  now  arose 
whether  the  causation  of  development  depended  in  this  case 
upon  the  withdrawal  of  water,  or  whether  the  swelling  of  the 
egg,  when  it  was  replaced  in  normal  sea-water,  had  something 
to  do  with  the  result.  With  this  object  in  view,  unfertilized 
sea-urchin  eggs  were  placed  for  a  long  time  in  slightly  hyper- 
tonic sea-w^ater,  viz.,  93  c.c.  of  sea-water+7  c.c.  2J  m  NaCl 
solution.  Now  it  turned  out  that  some  eggs  began  to  segment 
in  that  solution  and  that  a  few  reached  an  early  blastula  stage 
and  swam  about.  That  they  developed  no  farther  is  due  to  the 
fact  that  even  so  weak  a  hypertonic  solution  harmed  the  eggs 
when  they  remained  in  it  too  long.  Hence  the  experiment 
showed  that,  so  far  as  the  developmental  effect  of  the  hyper- 
tonic solution  is  concerned,  it  is  unnecessary  to  replace  the 
eggs  in  normal  sea-water.  The  latter  is  only  necessary  if  we 
wish  to  maintain  the  eggs  in  their  complete  vitality  and  to  allow 
them  to  develop  into  plutei. 

At  that  time  I  was  inclined  to  assume  that  the  effect  of 
the  hypertonic  solution  consisted  in  the  liquefaction  of  the 

»  The  eggs  of  different  females  show  a  different  degree  of  sensitiveness  to  the 
treatment  with  hypertonic  sea-water.  The  eggs  of  S.  purpuratus  are  more  often 
refractory  than  those  of  Arbacia. 


62      Artificial  Parthenogenesis  and  Fertilization 

nuclear  membrane  and  other  nuclear  constituents.^  Such  a 
liquefaction  must  of  course  occur  at  each  nuclear  division; 
but  I  believe  that  it  is  only  an  indirect  result  of  chemical 
processes  set  up  in  the  egg  by  the  hypertonic  solution,  and  not  a 
direct  effect  of  the  hypertonic  solution. 

Now  since  the  hypertonic  solution  works  entirely  in  virtue 
of  its  osmotic  pressure,  and  since  the  osmotic  pressure  depends 
only  upon  the  number  of  molecules  or  ions  in  the  unit  volume 
of  the  solution,  and  not  upon  the  chemical  nature  of  the  mole- 
cules or  ions,  all  isosmotic  solutions  should  be  equalty  effective 
in  causing  the  unfertilized  egg  to  develop.  This  is  approxi- 
mately true  (see  also  chap,  xiii),  except  that  solutions  containing 
poisonous  salts,  e.g.,  copper  salts  or  others,  cannot  be  used  for 
this  purpose. 

The  closer  the  hypertorfic  solution  approaches  sea-water  in 
its  composition,  the  less  harmful  become  the  secondary  effects 
of  the  hypertonic  solution.  For  this  reason  I  generally  use  for 
the  preparation  of  the  hypertonic  solution  sea-water  whose 
concentration  has  been  raised  to  the  desired  pitch  by  the  addi- 
tion of  a  suitable  amount  of  a  2J  m  NaCl,  or  Ringer,  solution. 
The  minimal  concentration  in  which  different  hypertonic  solu- 
tions are  effective  varies  somewhat  with  the  different  substances 
under  consideration. 

2;  My  next  experiments  aimed  at  determining  whether  the 
results  here  detailed  are  only  a  peculiarity  of  sea-urchin  eggs 
or  whether  artificial  parthenogenesis  can  be  produced  in  the 
eggs  of  all  animals.  In  so  doing  it  was  at  first  immaterial  to 
me  through  what  method  the  activation  of  the  egg  succeeded, 
so  long  as  it  was  only  possible  to  cause  the  unfertilized  eggs  to 
develop.  But  the  experiments  must  be  briefly  mentioned  here, 
since  the  experience  obtained  thereby  had  an  influence  upon  the 
further  development  of  this  line  of  research.  I  had  already  been 
able  to  show  in  the  3^ ear  1900  that  the  eggs  of  a  marine  worm, 

I  Loeb,   Am.  Jour.  Physiol.,  IV,  178,  1900. 


Osmotic  Parthenogenesis  1)3 


Chaetopterus,  can  be  caused  to  develop  by  the  addition  of  acid  or 
of  any  potassium  salt  to  the  sea-water  without  raising  its  osmotic 
pressure.^ 

Later  Neilson  and  I  showed  that  the  unfertiHzed  eggs  of  the 
starfish  (Asterias  forbesii)  can  be  made  to  develop  into  larvae  by 
any  inorganic  acid,  i.e.,  by  hydrions. 

In  collaboration  with  Fischer  I  discovered  that  the  addition 
of  a  calcium  salt  to  the  sea-water  causes  the  development  of  the 
unfertilized  eggs  of  a  marine  worm,  Amphitrite.-  Later  on  we 
will  return  to  these  investigations. 

1  Loeb,  Am.  Jour.  Physiol.,  IV,  423,  1901. 

2Loeb,  Fischer,  and  Neilson,  "Weitere  Versuciie  ueber  kuustliche  Partheno- 
genese,"   PflUger's  Archiv,  LXXXVII,  594,   1901. 


VIII 

THE   IMPROVED   METHOD   OF   ARTIFICIAL   PARTHENO- 
GENESIS  IN  THE  SEA-URCHIN   EGG 

The  first  method  of  producing  larvae  from  the  unfertilized 
egg  of  the  sea-urchin  by  a  mere  increase  of  osmotic  pressure 
only  sufficed  to  show  that  the  mysterious  complex  "living 
spermatozoon"  might  be  replaced  by  well-known  physico- 
chemical  agencies.  It  did  not  lend  itself  well  to  a  physico- 
chemical  analysis,  since  the  method  was  not  always  reliable. 
We  have  already  mentioned  that  with  the  Californian  sea- 
urchin  S.  purpuratus  the  method  worked  only  with  the  eggs  of 
a  small  percentage  of  females  and  even  the  eggs  of  different 
females  of  Arbacia  did  not  yield  equally  well  to  this  method. 
For  a  further  investigation  of  the  nature  of  the  process  of 
fertilization  this  method  was  therefore  very  unsatisfactor}-. 

There  were  other  reasons  which  indicated  that  a  better 
method  for  artificial  parthenogenesis  was  needed.  The  larvae 
of  normally  fertilized  eggs  rise  to  the  surface  of  the  water;  they 
are  pelagic.  The  larvae  produced  by  means  of  a  hypertonic 
solution  rarely  or  never  rose  to  the  surface  of  the  sea-water, 
but  swam  at  the  bottom  of  the  dish.  And  finally,  the  eggs 
form,  upon  the  entrance  of  a  spermatozoon,  the  fertilization 
membrane.  The  unfertilized  eggs  which  developed  after  treat- 
ment with  a  hypertonic  solution  never  formed  a  characteristic 
fertilization  membrane,  but  only  a  fine  gelatinous  film  instead 
of  the  clear  fertilization  membrane. 

This  led  me  to  think  that  the  osmotic  activation  of  the  egg 
was  only  an  incomplete  imitation  of  the  fertilization  process, 
and  that  the  fertilization  by  the  spermatozoon  perhaps  dei)end- 
ed  not  upon  a  single  chemical  agent,  })ut  upon  a  combination 
of  two  or  more  which  were  only  fortuitously  coml)ined  in  the 

65 


66      Artificial  Parthenogenesis  and  Fertilization 

spermatozoon.  This  idea  proved  to  be  correct.  I  had  just 
l^reviously  found  different  esters  to  be  especially  active  in  helio- 
tropic  experiments,  and  now  I  tried  the  effect  of  ethyl  acetate 
upon  the  unfertilized  eggs  of  S.  purpuratus. 

1.  It  turned  out  that  if  these  eggs  are  placed  in  sea-water 
to  which  a  little  ethyl  acetate  has  been  added,  they  form  a 
typical  fertilization  membrane  and  begin  to  divide  when 
replaced  in  normal  sea-water.  As  long  as  the  eggs  were  left 
in  the  sea-water  that  contained  ethyl  acetate,  they  formed  no 
mernbrane;  further,  they  lost  the  power  to  form  a  membrane 
if  the}^  remained  too  long  in  such  sea-water.  However,  they 
did  form  a  membrane  if  they  w^ere  exposed  to  the  ethyl  acetate 
in  sea-water  for  not  more  than  a  couple  of  minutes  and  were 
then  replaced  in  normal  sea-water.  Eggs  treated  in  this  manner 
all  formed  a  perfectly  normal  nuclear  spindle  after  an  hour, 
and  began  to  divide.  As  a  rule  they  did  not  develop  into 
larvae;  on  the  contrary,  the  eggs  went  to  pieces  in  less  than 
twenty-four  hours  (at  about  19°  C.)  without  reaching  the 
blastula  stage.  But  the  following  result  was  extremely  sur- 
prising. If  the  eggs  of  Strongylocentrotus  were  exposed  for  two 
hours  to  hypertonic  sea-water,  often  only  a  fraction  of  1  per 
cent  of  the  eggs  began  to  segment.  If,  however,  a  part  of  these 
eggs  were  subsequently  treated  with  ethyl  acetate  long  enough 
to  cause ,  the  formation  of  a  membrane  upon  transference 
to  normal  sea-water,  the  majority  of  the  eggs  developed  and 
many  in  quite  a  normal  manner.  In  the  latter  case  segmenta- 
tion followed  its  normal  course  and  at  normal  speed,  and  some 
of  the  larvae — probably  those  arising  from  normally  segmented 
eggs — rose  to  the  surface  of  the  water.  Here  then  we  had  a 
much  more  precise  imitation  of  the  process  of  fertilization.^ 

2.  The  next  step  was  to  determine  whether  this  depended 
upon  a  specific  action  of  the  ester  or  of  one  of  its  hydrolytic 

1  Loeb,  "On  an  Improved  ISIethod  of  Artificial  Parthenogenesis,"  University 
of  California  Publications,  Pliysiology,  II,  83,  1905. 


Improved  Method  of  Artificial  Parthenogenesis    67 


products.  It  turned  out  that  the  latter  was  the  case,  and  that 
any  monobasic  fatty  acid,  formic,  acetic,  propionic,  butyric, 
valerianic,  etc.,  induced  membrane  formation. 

If  the  unfertilized  eggs  of  S.  purpuratus  are  placed  in  50  c.c. 
of  sea-water+2.8  c.c.  N/10  butyric  acid  at  15°  C.  and  left  in 
this  solution  for  one  and  one-half  to  two  and  one-half  minutes, 
all  the  eggs  form  membranes  when  replaced  in  normal  sea-water. 
If  they  are  transferred  earlier  from  the  acid  to  normal  sea- 
water,  they  form  no  membrane;  nor  do  the  eggs  form  a  mem- 
brane after  remaining  too  long  in  the  acid,  because  the  acid 
injures  the  eggs. 

The  lower  monobasic  fatty  acids,  formic,  acetic,  propylic, 
valerianic,  and  capronic  acids,  behave  like  butyric  acid,  i.e., 
when  these  acids  are  added  to  sea-water  the  eggs  do  not  form 
membranes  while  thej^  are  in  the  acidulated  sea-water,  but  only 
after  they  are  transferred  to  normal  sea-water.  If,  however, 
the  higher  monobasic  fatty  acids  are  used,  e.g.,  heptylic,  capry- 
lic,  nonylic,  and  caprinic  acids,  the  eggs  form  a  fertilization 
membrane  while  they  are  in  the  sea-water  containing  the  acid. 
Carbonic  acid  behaves  hke  the  higher  fatty  acids.  The  differ- 
ent behavior  of  these  acids  finds  its  explanation  in  the  fact  that 
a  high  concentration  of  hydrogen  ions  in  the  surroundhig  solu- 
tion inhibits  the  membrane  formation.  We  shall  come  back  to 
this  point  later  on. 

Hence  if  one  of  these  lower  monobasic  fatty  acids  or  car- 
bonic acid  penetrates  into  the  egg,  it  acts  like  ethyl  acetate  in 
producing  membrane  formation.  If  now  the  unfertilized  eggs 
of  Strotigylocentrotus  were  first  placed  for  two  hours  in  hyper- 
tonic sea-water  and  then  treated -with  a  monobasic  fatty  acid, 
i.e.,  put  for  one  and  one-half  to  two  and  one-half  minutes  at 
15°  C.  into  50  c.c.  of.  sea-water+2.8  c.c.  N/10  butyric  acid, 
so  that  all  formed  membranes  upon  transference  to  normal 
sea-water,  all  the  eggs,  or  the  majority  of  them,  developed 
into  larvae;   while,  as  I  have  said,  less  than  1  per  cent  of  the 


68       Artificial  Parthenogenesis  and  Fertilization 

eggs  of  the  same  female  developed  when  treated  with  hyper- 
tonic sea-water  alone. ^ 

3.  The  next  question  which  arose  was  whether  the  fatty 
acid  starts  the  development  directly,  or  whether  its  action  is 
only  limited  to  the  setting-off  of  the  process  of  membrane 
formation;  in  that  event,  this  process,  formerly  regarded  as 
something  very  secondary,  would  be  established  as  the  essential 
factor  in  development.  The  question  was  answered  in  the 
latter  sense,  as  the  following  facts  show.  If  the  unfertilized 
eggs  of  the  sea-urchin  S.  purpuratus  are  placed  for  one  and  one-^ 
half  to  two  and  one-half  minutes  at  15°  C.  in  50  c.c.  of  sea-water 
+2.8  c.c.  of  a  N/10  monobasic  fatt}^  acid,  all  the  eggs  form 
a  membrane  upon  transference  to  ordinary  sea-water.  This 
result  is  so  constant  that  I  have  only  rarely  found  an 
exception  to  it.  But  if  the  eggs  are  removed  a  little  earlier  from 
the  acid  to  normal  sea-water,  one  can  find  a  period  of  time  in 
which  no  longer  all  the  eggs  but  only  some  of  them  form 
membranes.  It  will  be  found  that  only  such  eggs  as  have 
formed  membranes  develop,  if  they  are  subsequently  or 
previously  treated  with  hypertonic  sea- water. ^  Membrane 
formation  is  therefore  the  deciding  condition  for  development. 

A  further  proof  is  afforded  by  the  following  fact.  We  shall 
see  in  the  following  chapters  that  any  substance  which  causes 
haemolysis  also  calls  forth  membrane  formation,  e.g.,  saponin, 
bile  salts,  hydrocarbons,  ether,  etc.  No  matter  by  what 
means  the  membrane  formation  has  been  called  forth,  it  induces 
the  development  of  the  egg,  provided  that  the  eggs  are  taken  out 
of  these  solutions  at  the  proper  time.  If  such  eggs  which 
possess  a  membrane  are  afterward  subjected  to  treatment 
with  hypertonic  sea-water  they  develop  like  fertilized  eggs. 

1  Loeb,  "On  an  Improved  Method  of  Artificial  Parthenogenesis,"  2d  and  3d 
communications.  University  of  California  Publications,  Physiology,  II,  1905;  Un- 
tersuchungen  zur  kiinstlichen  Parthenogenese,  pp.  322  and  329,  Leipzig,  1906. 

2  The  beginner  must  bear  in  mind  that  the  membrane  occasionally  adheres 
closely  to  the  egg  and  that  occasionally  the  process  of  membrane  formation  is  not 
completed.     However,  such  eggs  can  develop. 


Improved  Method  of  artificial  Parthenogenesis    (39 

Hence  membrane  formation  is  essential  for  development,  and 
it  is  immaterial  how  the  membrane  formation  has  been  pro- 
duced; except  that  the  membrane  formation  induced  by  a 
fatty  acid  injures  the  egg  less  than  that  provoked  by  most  of 
the  other  substances. 

4.  In  the  experiments  thus  far  discussed  the  treatment  with 
the  hypertonic  solution  preceded  the  artificial  membrane  forma- 
tion with  the  fatty  acid.  Fertilization  with  sperm,  however, 
begins  with  membrane  formation;  and  hence  it  was  natural  to 
find  out,  whether  in  artificial  parthenogenesis  also,  membrane 
formation  by  means  of  a  fatty  acid  might  not  be  applied  as  the 
first  step. 

The  unfertilized  eggs  were  therefore  subjected  to  treatment 
with  a  fatty  acid  until  they  all  formed  membranes.  Ten  to 
twenty  minutes  later  they  were  placed  in  hypertonic  sea-water. 
It  turned  out  that  in  this  order  of  events  the  eggs  of  S.  purpur- 
atus  need  remain  in  the  hypertonic  solution  only  a  relatively 
short  time,  some  thirty  to  sixty  minutes  at  about  J5°C.^ 
The  development  of  the  eggs  is  nearly  identical  with  that  evoked 
by  sperm,  and  practically  all  the  eggs  develop;  provided  that 
the  details  of  the  method  are  correctly  carried  out. 

The  procedure  for  the  chemical  activation  of  the  unferti- 
lized egg  of  S.  purpuratus  is  therefore  as  follows.  The  eggs 
are  placed  in  50  c.c.  of  sea-water +2 . 8  c.c.  of  N/IO  butyric  acid 
(which  must  be  thoroughly  mixed  beforehand).  At  15°  C. 
a  portion  of  the  eggs  is  transferred  after  one  and  one-half, 
two  and  one-half,  and  three  or  four  minutes  to  200  c.c.  of  sea- 
water  which  had  previously  been  made  ready  for  this  purpose. 
In  one  or  ail  of  the  dishes  all  the  eggs  form  a  normal  fertilization 
membrane. 

It  may  be  observed,  by  the  way,  that  too  many  eggs  must  not 
be  put  into  the  acid  sea-water,  since  otherwise  the  mass  of  acid 

1  The  eggs  of  Arbacia  need  remain  in  the  hypertonic  solution  only  about  20 
minutes  at  a  temperature  of  22°  C. 


70      Artificial  Parthenogenesis  and  Fertilization 


is  not  sufficient.  It  is  also  necessary  to  collect  the  eggs  into 
a  heap  by  gentle  rotation  of  tlie  dish  before  transferring  them 
to  normal  sea-water,  so  that  they  can  be  transferred  in  a 
pipette  with  as  little  acid  as  possible.  This  gentle  agitation 
also  prevents  the  eggs  from  sticking  to  the  glass. 

After  the  eggs  are  transferred  from  the  acid  to  normal  sea- 
water,  they  should  not  be  placed  at  once  in  the  hypertonic 
sea-water,  but  only  after  from  fifteen  or  twenty  minutes,  or 
even  later  still.  The  h>T)ertonic  sea-water  used  in  this  case 
is  a  mixture  of  50  c.c.  of  sea-water +8  c.c.  2 J  m  NaCi.^  From 
this  they  are  transferred  to  normal  sea-water  after  from  thirty 
to  sixty  minutes  at  15°  C,  at  intervals  of  from  two  and  one- 
half  to  five  minutes.  After  transference  to  normal  sea-water, 
those  eggs  which  have  been  just  long  enough  in  the  hypertonic 
solution  begin  to  segment  and  develop.  Usually  only  two 
astrospheres  or  centrosomes  are  formed  by  this  method. 
When  the  length  of  exposure  is  correct,  the  first  division  is  a 
cleavage  of  the  egg  into  two  cells  as  in  normal  fertilization, 
although  the  cleavage  in  the  first  division  is  often  somewhat 
asymmetrical.  That  is  probably  a  result  of  treating  the  egg 
with  hypertonic  sea-water.  This  anomaly,  however,  only 
presents  itself  in  the  first  division  and  has  no  further  influence 
on  the  development.  All  the  eggs  that  divide  into  two  cells 
develop  into  apparently  normal  larvae,  while  eggs  which  go 
into  more  than  two  cells  at  one  division  develop  into  crippled 
larvae  and  most  of  them  die  in  or  before  the  gastrula  stage.  - 
This  latter  abnormal  kind  of  development  is  obtained  regularl}^ 
if  the  eggs  remain  too  long  in  the  hypertonic  solution  and  an 
over-exposure  of  only  a  few  minutes  may  produce  this  fatal 
effect.  This  shows  how  necessary  it  is  that  the- eggs  be  trans- 
ferred from  the  hypertonic  solution  to  normal  sea-water  at  the 
right  time.2 

•  a  mixture  of  2^  m  NaCl  +KC1  +CaCL  in  the  proportions  in  which  these 
salts  exist  in  the  sea-water  is  still  better  than  2J  m  NaCl,  since  it  is  less  injurious. 

"  This  time  varies  somewhat  for  the  different  eggs  even  of  the  same  female. 


Improved  Method  of  Artificial  Parthenogenesis    71 

It  is,  moreover,  necessary  that  not  too  many  eggs  be  put 
into  one  bowl  of  hypertonic  sea-water,  since  otherwise  a  mutual 
struggle  for  oxygen  takes  place.  The  eggs  should  also  be  kept 
in  shallow  dishes  so  that  the  layer  of  water  which  covers  them 
may  not  be  too  deep  and  the  diffusion  of  oxygen  from  the  air  to 
the  eggs  not  too  greatly  hindered.  I  usually  cover  the  bowls 
loosely  with  a  glass  plate. ^ 

I  have  since  tried  the  same  method  with  success  on  the  eggs 
of  Arbacia  at  Woods  Hole.  As  was  to  be  expected,  the  quanti- 
tative data  of  the  method  are  a  little  different  in  this  case  from 
those  found  in  *S.  purpuratus.  The  eggs  were  put  into  a  mix- 
ture of  50  c.c.  sea-water +2  c.c.  N/10  butyric  acid  for  from  one 
and  one-half  to  three  minutes.  When  transferred  to  sea-water 
they  did  not  form  a  conspicuous  fertilization  membrane,  as  did 
the  eggs  of  S.  purpuratus  under  the  same  circumstances,  but 
only  a  fine  gelatinous  layer  which  was  not  easily  visible.  Ten 
or  fifteen  minutes  later  the  eggs  were  transferred  to  hypertonic 
sea-water  (50  c.c.  sea-water +8  c.c.  2 J  m  NaCl).  In  this  solu- 
tion the  eggs  remained  at  a  temperature  of  about  23°  onh'  from 
seventeen  and  one-half  to  twenty-five  minutes,  after  whicJi 
they  were  transferred  to  normal  sea-water.  Since  the  time 
during  which  the  eggs  must  remain  in  the  hj'pertonic  solution 
varies  for  the  individual  eggs  and  since  a  slight  over-exposure 
in  the  hypertonic  solution  seriously  injures  the  eggs,  it  is  advis- 
able to  transfer  the  eggs  not  all  at  the  same  time  but  at  intervals, 
e.g.,  after  seventeen  and  one-half,  twenty,  twenty-two  and 
one-half,  and  twenty-five  minutes.  Eggs  which  were  exposed 
longer  than  twenty-five  minutes  to  the  hypertonic  solution  did 
not  develop. 

The  same  method  has  been  applied  successfully,  among 
others  at  Naples  by  Herbst,  Warburg,  and  by  Meyerhof,  and 
at  Plymouth  by  Shearer  and  his  pupils. 

1  other  factors  which  have  to  bo  considered  iu  this  method  will  be  mentioned 
in  the  next  chapter. 


IX 

THE  EFFECT  OF  ARTIFICIAL   MEMBRANE  FORMATION 

ON  THE  EGG 

We  shall  see  later  that  for  the  eggs  of  starfishes  and  various 
annelids  (Polynoe  and  Thalassema)  the  induction  of  artificial 
mem])rane  formation  is  sufficient  to  cause  some  of  the  unferti- 
lized eggs  to  develop  into  larvae.  According  to  the  experi- 
ments of  Herbst,  to  which  we  shall  return  later,  something 
similar  appears  to  be  the  case  exceptionally  with  the  eggs  of 
the  Neapolitan  sea-urchins  also.  Neither  the  eggs  of  the 
Calif ornian  sea-urchins,  nor  those  of  Arhacia  at  Woods  Hole,  do 
as  a  rule  develop  into  larvae  if  artificial  membrane  formation 
alone  is  produced  in  them.  They  do,  however,  ^vithout  excep- 
tion exhibit  the  initial  changes  of  development  after  such 
treatment.  It  depends  upon  the  temperature  how  far  they  will 
develop.  If  the  temperature  is  very  low  (from  2°  to  5°)  the  eggs 
divide  very  slowly  and  regularly,  and  a  few  may  develop  into 
swimming  larvae,^  although  they  do  not  survive  the  blastula 
stage.  If  the  temperature  is  a  little  higher,  some  6°  to  8°,  the 
eggs  go  into  the  two-,  four-,  eight-,  and  even  sixteen-cell  stage, 
but  no  farther.  At  higher  temperatures  still,  say  15°  to  18°  C, 
a  formation  of  astrospheres  and  nuclear  division  takes  place; 
but  then  the  development  comes  to  a  standstill  and  the  eggs  do 
not  divide,  but  slowly  disintegrate. 

The  fact  that  the  egg  can  develop  at  a  low  temperature  to 
the  blastula  stage,  and  that  nuclear  division  and  cell  division 
begin  always  after  the  membrane  formation,  proves  that  this 
process  suffices  to  set  the  whole  apparatus  of  development  in 
motion.     The  subsequent  treatment  with  a  hypertonic  solution 

1  Loeb,  U Titer suchungen  ueber  kanstliche  Parthenogenese,  p.  4i>0;  Biochem. 
Zeitschr.,  I.  203,  1906. 

73 


74      Artificial  Parthenogenesis  and  Fertilization 


has,  therefore,  nothing  to  do  with  the  activation  of  the  egg. 
The  question  arises :   What  is  its  function  ? 

We  receive  an  answer  to  this  question  when  we  observe 
what  becomes  of  the  eggs   at   room   temperature  after   the 


Fig.  20 


Fig.  19 

Figs  19  and  20.— Beginning  of  the  process  of  disintegration  after  the  arti- 
ficial membrane  formation  in  the  egg  of  S.  purpuratus.  Small  droplets  appear  at 
the  equator  of  the  elongated  egg. 

membran3  formation.  In  this  case  the  centrosomes  and  two 
astrospheres  are  formed  and  the  nucleus  may  divide,  but  then  a 
process  of  disintegration  begins  in  the  egg.  Small  droplets 
appear  usually  on  one  side  of  the  egg  (Figs.  19,  20).  One  gains 
the  impression  that  these  droplets  begin  to  be  extruded  at  the 


Fig.  21 


Fig.  22 


Figs.  21  and  22. — Eggs  of  the  same  lot,  showing  more  plainly  that  the  droplet 
formation  and  disintegration  begin  in  the  plane  of  cell-division. 

time  of  the  first  cell  division.  This  is  obvious  from  Figs. 
21  and  22,  where  the  droplets  appear  in  the  plane  of  segmenta- 
tion. In  Figs.  19  and  20  they  appear  in  the  equatorial  plane 
of  eggs  which  are  not  segmented,  but  which  show  the  elonga- 
tion in  the  direction  of  the  two  poles. 


Effect  of  Artificial  Membrane  Formation 


75 


\Mien  the  disintegration  has  once  commenced,  it  continues 
until,  after  a  certain  time  which  varies  inversely  with  the 
temperature,  the  whole  egg  is  disintegrated.  Fig.  23  represents 
an  egg  a  few  hours  after  artificial  membrane  formation,  in 


Fig.  23 


Fig.  24 


Fig.  26 


Fig.  25 

Ftgs  23-26  —Disintegration  of  the  sea-urchin  egg  after  artificial  membrane 
formation,  if  it  is  not  subSiitted  to  treatment  with  a  hypertonic  solution  or  an 
inhibition  of  oxidations. 

which  the  nuclear  spindle  is  visible.  It  did  not  develop  farther, 
but  a  httle  later  a  few  clear  drops  (Fig.  24),  resembling  polar 
bodies,  appeared  to  exude  from  the  egg;  I  suspect  that  this 
extravasation  of  drops  took  place  in  the  equatorial  plane.  This 
disintegration  then  proceeds  farther  (Fig.  25),  until  finally  the 
egg  is  almost  completely  destroyed  (Fig.  26). 


76      Artificial  Parthenogenesis  and  Fertilization 


While  Figs.  24-26  give  a  picture  of  the  decay  of  an  egg  at 
room  temperature,  the  start  of  disintegration  at  a  somewhat 
lower  temperature  is  represented  in  Figs.  27-31.  The  eggs  were 
kept  at  a  temperature  between  5°  to  10°  C.  after  membrane 
formation  with  butyric  acid.  Many  divided  and  the  disinte- 
gration began  later.     Drawings  were  made  of  a  series  of  differ- 


Fig.  27 


Fig.  28 


Fig.  29 


Fig.  30 


Fig.  31 


Figs.  27-31. — Slow  disintegration  of  the  egg  of  the  sea-urchin  at  low  tem- 
perature.    The  egg  can  reach  the  eight-ceU  stage  under  such  conditions. 

ent  eggs  in  incipient  disintegration.  In  all  cases  this  started 
with  the  formation  of  small  drops,  usually  in  the  plane  of 
division;  the  result  is  always  similar  to  that  shown  in  Fig.  26. 
We  now  can  answer  the  question,  what  is  the  role  of  the 
subsequent  treatment  of  the  egg  with  the  hypertonic  solution: 
it  serves  the  purpose  of  preventing  the  disintegration  which 
begins  at  the  time  of  the  first  cell  division.  The  conditions 
for  this  action  of  the  hypertonic  solution  will  be  considered  in  a 
subsequent  chapter. 


Effect  of  Artificial  Membrane  Formation         77 


It  is  of  interest  to  add  that  fertilized  eggf>  can  also  be  caused 
to  show  this  process  of  droplet  formation  during  the  first  seg- 
mentation, namely  if  they  are  caused  to  segment  in  an  abnormal 
solution.  It  depends  upon  the  degree  of  abnormality  whether 
or  not  such  eggs  can  afterward  develop  into  normal  larvae. 
When  unfertilized  eggs  of  S.  purpuratus  were  exposed  for  about 
two  hours  to  a  hypertonic  solution  and  fertilized  with  sperm 
immediately  after  they  were  taken  out  of  the  solution  they  were 
able  to  develop,  but  the  first  segmentation  occurred  often  with 
droplet  formation  in  the  plane  of  division.  AVhen  they  were 
fertilized  after  they  had  been  in  the  normal  sea-water  for  some 
time  they  segmented  normally. 

While  the  artificial  membrane  is  enough  to  initiate  the  act 
of  development,  something  is  abnormal  in  the  egg  in  so  far  as  the 
process  of  cell  division  leads  to  its  destruction.  We  should 
therefore  expect  that  if  we  inliibit  the  process  of  cell  division, 
the  eggs  would  not  perish.     This  conclusion  is  correct. 

It  has  been  mentioned  that  cell  division  can  be  prevented 
in  the  fertilized  sea-urchin  egg  by  depriving  it  of  ox\'gen  or 
stopping  oxidation  by  certain  poisons,  such  as  KCN.  We  will 
now  proceed  to  show  that  if  we  deprive  the  sea-urchin  egg  of 
oxygen  after  artificial  membrane  formation,  or  put  it  in  sea- 
water  with  a  little  KCN' or  chloral  hydrate,  we  then  inhibit  the 
disintegration  described  above. ^  Membrane  formation  was  pro- 
duced in  the  eggs  of  a  sea-urchin  by  treating  them  with  butyric 
acid.  The  eggs  were  then  divided  into  three  portions.  One 
remained  exposed  to  the  air,  a  second  was  placed  in  a  flask 
through  which  was  passed  a  stream  of  pure  oxygen,  the  third 
in  a  flask  of  sea-water  out  of  which  practically  all  air  had  been 
driven  by  a  stream  of  hydrogen,  and  in  which  the  current  of 
hydrogen  passed  through  the  vessel  during  the  whole  of  the 
experiment.     The  eggs  which  had  been  exposed  to  oxygen  in 

1  Loeb,     Untersuchungen    ueber    kunstliche    Parthenogentse,    p.    483;     Biochcm, 
Zeitschr.,  I.  192,  1906. 


78      Artificial  Parthenogenesis  and  Fertilization 


the  first  two  vessels  had  all  formed  nuclear  spindles  at  the  right" 
time,  and  began  to  disintegrate  after  four  or  five  hours.  After 
ten  hours  none  of  these  eggs  could  be  induced  to  develop  bj^ 
treatment  with  hypertonic  sea-water.  On  the  other  hand,  the 
eggs  which  had  been  in  the  current  of  hydrogen  had  formed  no 
spindle  and  were  perfectly  intact  when  disconnected  from  it. 
They  could  still  be  induced  to  develop  by  treatment  with  hyper- 
tonic sea-water.  Even  after  remaining  twenty-four  hours  in 
the  stream  of  hydrogen  the  eggs  maintained  their  shape  intact. 
Just  the  same  sort  of  results  were  obtained  upon  the  addition  of 
a  little  KCN  to  the  sea-water,  by  which  means  the  development 
was  arrested.  By  stopping  development  (as  a  result  of  inhibit- 
ing oxidations)  the  eggs  are  kept  from  that  disintegration  after 
membrane  formation  to  which  they  would  be  condemned  at 
room  temperature.  Hence  we  must  come  to  the  conclusion 
that  membrane  formation  does  indeed  initiate  development, 
but  that  it  leaves  the  egg  in  a  condition  in  which  the  cell  divi- 
sion becomes  fatal  to  it.  If  we  prevent  cell  division  the  egg 
survives.^ 

This  conclusion  is  supported  by  the  observation  that  if  the 
development  of  the  eggs  after  artificial  membrane  formation  be 
stopped  for  several  hours,  they  are  then  able  to  develop  even  at 
room  temperature,  and  not  only  to  blastulae  but  also  to  plutei.^ 
I  first  became  aware  of  this  fact  when  I  put  eggs  after  mem- 
brane formation  (with  butyric  acid)  into  sea-water  from  which 
the  air  had  been  displaced  by  a  current  of  hydrogen.  At 
various  intervals  eggs  were  replaced  in  normal  sea-water  from 
that  lacking  oxygen.  It  turned  out  that  eggs  which  at  15°  C. 
had  been  less  than  three  hours  in  the  sea-water  that  lacked 
oxygen  all  disintegrated  after  the  return  to  normal  sea-water; 
but  that  of  the  eggs  which  had  been  three  hours  or  a  Uttle  longer 

1  Chloral  hydrate  acts  in  the  same  way,  although  It  does  not  diminish  the  rate 
of  oxidation ;  but  it  inhibits  the  developmental  process  and  cell  division  and  thus 
prevents  the  fatal  disintegration  of  the  egg. 

2Loeb,  op.  cit. 


Effect  of  Artificial  Membrane  Formation         79 

in  the  sea-water  poor  in  oxygen,  some  (about  1  per  cent)  devel- 
oped to  perfectly  normal  plutei  after  they  were  transferred  to 
normal  sea-water.  Experiments  with  potassium  cyanide  gave 
the  same  results.  Unfertilized  eggs  in  which  a  but3Tic  acid 
membrane  had  been  formed  were  placed  in  50  c.c.  of  sea-water 
-|-1  c.c.  of  a  1  per  cent  KCN  solution.  Here  the  concentration 
was  forty  times  as  large  as  is  necessary  for  the  inhibition  of 
the  division  of  the  fertilized  egg  and  for  the  prevention  of 
the  disintegration  process.  Some  of  the  eggs  were  trans- 
ferred to  normal  sea-water  after  18,  30,  45,  65,  and  85 
minutes.  Of  the  eggs  placed  in  normal  sea-water  after  45 
and  65  minutes  a  small  number,  about  5  per  cent,  developed. 
In  this  case,  however,  the  development  did  not  start  till  fourteen 
hours  after  the  eggs  were  taken  out  of  the  sea-water  that  con- 
tained the  potassium  cyanide.  I  suppose  that  several  hours 
are  necessary  for  all  the  hj^drocyanic  acid  to  disappear  from 
the  egg.  This  conjecture  is  supported  by  the  fact  that  only 
those  eggs  developed  which  were  placed  after  treatment  with 
potassium  cyanide  in  flat  watch  glasses  in  which  they  were 
covered  only  by  a  shallow  layer  of  water,  and  in  which  therefore 
the  evaporation  of  the  HCN  could  proceed  ciuickly.  The 
development  of  the  eggs  w^as  also  favored  by  passing  a  stream 
of  oxygen  through  the  watch  glasses  for  a  minute.  But  similar 
results  could  also  be  obtained  with  smaller  quantities  of  KCN. 
Thus,  in  one  experiment,  the  eggs  were  placed  after  artificial 
membrane  formation  in  50  c.c.  of  sea-water  to  which  1,  2,  4, 
and  8  c.c.  of  1/10  of  1  per  cent  KCN  had  been  added.  They 
remained  in  these  solutions  for  from  one  to  twenty-three  hours. 
A  few  of  the  eggs  which  had  been  transferred  from  the  solutions 
with  2  and  4  c.c.  of  1/10  of  1  per  cent  KCN  to  normal  sea-water 
after  from  three  to  seven  hours  developed  into  larvae. 

It  struck  me  in  these  experiments  that  the  development  of 
the  eggs  appeared  wonderfully  normal,  and  so  several  years  ago 
I   took  up  these  experiments  afresh.     In  order  to   convince 


80      Artificial  Parthenogenesis  and  Fertilization 


myself  that  membrane  formation  is  the  real  force  that  activates 
the  unfertilized  egg,  it  was  necessary  to  show  that  all  sea-urchin 
eggs  can  be  caused  to  develop  through  this  agent  alone. 

In  my  earlier  experiments  the  eggs  were  placed  in  the  potas- 
sium cyanide  sea-water  very  soon  after  membrane  formation. 
Now  this  is  in  great  part  answerable  for  the  fact  that  the  num- 
ber of  eggs  which  afterward  developed  into  larvae  remained  so 
small.  For  I  have  recently  found  that  if  the  eggs  are  placed  in 
this  solution  a  little  later  a  large  percentage  of  larvae  can  be 
obtained  and  in  many  experiments  all  the  eggs  developed  into 
larvae  after  transference  to  normal  sea-water.  In  this  experi- 
ment a  very  weak  solution  of  KCN  was  used,  viz.,  a  mixture  of 
50  c.c.  of  sea-water +  1  or  2  c.c.  1/20  of  1  per  cent  KCN. 

Membrane  formation  was  produced  in  the  eggs  of  a  sea- 
urchin  (*S.  purpuratus)  by  means  of  butyric  acid.  Some  of  the 
eggs  were  transferred  immediately  (i.e.,  two  minutes)  after 
membrane  formation  in  50  c.c.  of  sea-water +2  c.c.  1/20  of 
1  per  cent  KCN.  After  three,  four,  and  five  hours  the  eggs 
were  transferred  to  normal  sea-water  (after  they  had  been 
freed  from  KCN  by  thrice  washing  in  sea-water).  The  tem- 
perature was  11 . 5°  C.     Not  a  single  egg  developed  into  a  larva. 

A  second  portion  of  the  eggs  was  placed  in  the  sea-water 
and  potassium  cyanide  twenty  minutes  after  membrane  forma- 
tion. After  three  hours  some  of  these  eggs  were  transferred  to 
normal  sea-water  and  about  5  per  cent  developed  into  larvae. 
About  10  per  cent  and  20  per  cent  of  larvae  respectively  were 
produced  by  the  eggs  removed  from  the  cyanide  sea-water 
after  four  and  five  hours. 

A  third  portion  of  the  eggs  was  transferred  to  the  cyanide 
sea-water  forty-three  minutes  after  membrane  formation. 
Practically  all  of  the  eggs  transferred  to  normal  sea-water  after 
three  hours  developed  into  larvae.  The  eggs  that  remained 
longer  in  the  cyanide  were  injured  and  did  not  develop  so  well. 

Eggs    transferred    to    the    cyanide    sea-water    fifty-seven 


Effect  of  Artificial  Membrane  Formation         81 


minutes  after  membrane  formation  behaved  in  a  similar  manner. 
Others,  transferred  eighty-two  minutes  after  membrane  forma- 
tion, gave  few  larvae. 

These  experiments  were  repeated  quite  often,  but  the  results 
were  not  always  equally  good.  It  is  possible  that  the  eggs  of 
various  females  did  not  react  equally  well  to  this  method. 

In  the  more  recent  experiments  the  eggs  were  first  washed 
two  or  three  times  with  ordinary  sea-water  after  being  taken 
from  that  containing  potassium  cyanide. 

In  all  these  experiments  the  perfect  regularity  of  the  seg- 
mentation was  very  striking;  the  segmentation  is  almost  as 
good  as  in  eggs  fertilized  with  sperm.  This  is  an  important  fact, 
in  view  of  the  experiments  in  which  the  disintegration  of  the 
eggs  after  membrane  formation  is  prevented  by  hypertonic 
sea-water.  In  this  case  the  first  division  is  often  somewhat 
irregular.  The  experiments  with  potassium  cyanide  show 
that  this  irregularity  is  essentially  a.  secondary  effect  of  the 
hypertonic  salt  solution,  and  that  membrane  formation  alone 
leads  to  a  normal  segmentation. 

Secondly  it  must  be  remarked  that  in  these  experiments  tlie 
larvae  stayed  on  the  surface  of  the  sea-water  as  in  normal  devel- 
opment, and  gave  large  numbers  of  plutei.  The  shorter  the 
time  that  the  eggs  had  remained  in  the  potassium  cyanide 
solution,  the  larger  the  number  of  eggs  that  developed  into 
normal  plutei.  The  reason  is  obvious.  Membrane  formation 
sets  going  (or  accelerates)  the  chemical  reactions  which  deter- 
mine development.  If  we  interrupt  the  oxidations  in  such  an 
egg,  there  will  accumulate  in  it  decomposition  products  which 
ought  to  be  removed  by  oxidation.  I  suppose  that  this  accu- 
mulation of  decomposition  products  leads  to  the  injury  of  the 
egg,  which  is  shown  by  the  fact  that  the  longer  the  eggs  remain 
in  the  cyanide  solution,  the  more  their  vitality  is  imi)air('d. 
We  have  had  the  same  experience  with  sea-urchin  eggs  ferti- 
lized with  sperm,  which  also  lose  their  vitality  more  quickly 


82      Artificial  Parthenogenesis  and  Fertilization 


and  die  in  an  earlier  larval  stage  the  longer  they  have  been 
exposed  to  lack  of  oxygen  (or  to  KCN).  Eggs  which  remained 
in  the  cyanide  sea-water  only  two  or  three  hours  after  mem- 
brane formation  produced  plutei  in  relatively  greater  numbers 
than  those  that  remained  longer  in  this  solution  (see  chap.  iii). 

The  optimum  length  of  exposure  of  the  eggs  to  the  potassium 
cyanide  solution  depends  also  upon  the  concentration  of  KCN 
in  it.  In  a  mixture  of  50  c.c.  of  sea-water +2  c.c.  1/20  of  1  per 
cent  KCN,  three  hours  seem  to  represent  the  optimum  length 
of  exposure.  If  the  eggs  remain  only  two  hours  in  the  cyanide 
solution,  larvae  are  usually,  but  not  always,  obtained.  Thus, 
in  one  experiment  the  eggs  were  placed  two  hours  after  arti- 
ficial membrane  formation  in  50  c.c.  of  sea-water -f  2  c.c.  of  1/20 
of  1  per  cent  KCN.  Of  the  eggs  which  were  removed  from  thih 
solution  after  two  hours,  washed  twice  in  sea-water,  and  then 
transferred  to  normal  sea-water,  50  per  cent  developed  into 
good  larvae;  90  per  cent  of  those  removed  after  three  hours 
grew  into  larvae;  but  the  length  of  life  of  the  former  was  greater 
than  that  of  the  latter. 

The  method  of  treating  the  eggs  after  membrane  formation 
with  lack  of  oxygen  or  with  KCN  has,  however,  the  disad- 
vantage that  it  is  technically  very  difficult  and,  secondly, 
that  the  time  that  must  elapse  between  artificial  membrane 
formation  and  transference  to  the  potassium  cyanide  solution 
is  obviously  not  always  the  same.  I  have  always  found  that 
if  the  eggs  are  transferred  to  the  cyanide  solution  immediately 
after  membrane  formation,  none,  or  only  a  few,  of  the  eggs 
develop;  but  I  am  unable  to  say  how  long  after  membrane 
formation  one  ought  to  wait  before   placing  the  eggs  in  the 

cyanide  solution. 

These  experiments  prove  at  least  that  it  is  possible  to  sub- 
stitute for  the  corrective  influence  of  a  short  treatment  with  a 
hypertonic  solution  a  suppression  of  the  development  of  the  egg 
for  a  longer  period  of  time.     After  such  a  treatment  the  egg 


Effect  of  Artificial  Membrane  Formation         83 


seems  to  be  able  to  segment  without  disintegrating  in  the 
process.  While  the  eggs  are  without  oxygen  or  in  the  cyanide 
solution  changes  occur  in  the  eggs  which  allow  them  to  undergo 
cell  division  in  a  normal  way,  when  put  back  into  normal  sea- 
water.  While  this  method  is  theoretically  of  interest,  the 
method  of  treating  the  eggs  with  hypertonic  sea-water  after 
the  membrane  formation  is  for  practical  purposes  preferable, 
since  it  is  absolutely  reliable. 

The  experiments  thus  far  reported  show  therefore  that  the 
artificial  membrane  formation  starts  the  development  of  the 
sea-urchin  egg,  but  that  it  leaves  the  egg  in  a  sickly  condition 
which  causes  it  to  disintegrate  rapidly  at  room  temperature. 
In  order  to  make  such  eggs  normal,  they  must  undergo  a  second 
treatment:  they  must  either  be  exposed  for  a  short  time  to  a 
hypertonic  solution  or  for  a  somewhat  longer  time  to  normal 
sea-water  in  which  the  oxidations  of  the  egg  are  suppressed. 


X 

FURTHER  EXAMPLES  OF  THE  PROLONGATION  OF  THE 
LIFE  OF  THE  EGG  BY  LACK  OF  OXYGEN 

1.  It  is  rather  curious  that  lack  of  oxygen,  or  probably  the 
suppression  of  cell  division,  should  be  able  to  delay  the  disinte- 
gration with  which  the  egg  of  the  sea-urchin  is  threatened  after 
artificial  membrane  formation.  We  found  an  explanation  for 
this  phenomenon  in  the  fact  that  the  process  of  nuclear  or  cell 
division  is  critical  for  the  egg  in  danger  of  disintegration.  If 
one  suppresses  this  process  the  disintegration  is  markedh- 
retarded.  The  cell  division  can  be  suppressed  or  retarded  by 
the  suppression  of  oxidations  as  well  as  by  narcotics.^  We  also 
called  attention  to  the  fact  that  if  we  allow  normally  fertilized 
eggs  to  segment  in  abnormal  solutions,  the  process  of  cell  divi- 
sion is  also  accompanied  by  processes  of  disintegration  (droplet 
formation)  comparable  to  those  which  are  characteristic  for  the 
egg  after  artificial  membrane  formation.  We  should  therefore 
have  a  right  to  expect  that  if  we  put  fertilized  eggs  into  abnormal 
solutions,  their  lives  will  be  prolonged  if  we  inhibit  cell  division 
either  by  lack  of  ox>'gen  or  by  KCN  or  by  other  means  which 
inhibit  cell  division,  e.g.,  narcotics.  We  should  also  logically 
be  led  to  suppose  that  unfertilized  eggs  will  live  longer  in  such 
abnormal  solutions  than  fertilized  eggs,  simply  for  the  reason 
that  they  are  not  threatened  with  nuclear  or  cell  division. 

The  writer  has  made  a  series  of  observations  which  show 
that  quite  generally  unfertilized  eggs  suffer  less  in  abnormal 
solutions  than  fertilized  eggs;  and  that  fertilized  eggs  suffer 
less  rapidly  if  their  oxidations  are  suppresstnl. 

1  The  narcotics  suppress  or  retard  the  process  of  cell  division  and  ilevelopinenl 
without  diminishing  the  rate  of  oxidation  in  the  egg  noticeably.  Loeb  and  Waste- 
neys.  Jour.  Biol.  C/iem..-XIV.  518.  1913. 

85 


86      Artificial  Parthenogenesis  and  Fertilization 


2.  The  writer  pointed  out  in  1906  that  the  fertiUzed  egg 
of  the  sea-urchin  is  injured  much  more  rapidly  by  abnormal 
solutions  than  the  unfertilized  egg.  Fertilized  and  unfertilized 
eggs  of  the  same  purpuratus  female  were  put  into  a  neutral 
m/2  solution  of  NaCl.  From  time  to  time  specimens  of  these 
eggs  were  transferred  to  sea-water  to  test  whether  or  not 
they  were  normal.  The  fertilized  eggs  ceased  to  develop  into 
b!astulae  when  they  were  more  than  three  hours  in  the  m/2 
NaCl;  while  the  unfertilized  eggs  developed  normally  when 
sperm  was  added  after  they  had  been  in  the  NaCl  solution  for 
forty-eight  hours.  The  same  could  be  proved  for  solutions  of 
KCl,  CaCla,  MgCla,  and  other  salts.i 

When  we  put  fertilized  and  unfertilized  eggs  of  purpuratus 
into  a  mixture  of  NaCl+KCl  to  which  some  strong  base  has 
been  added,  the  fertilized  eggs  are  destroyed  much  more  rapidly 
than  the  unfertilized  eggs.  In  fact,  the  unfertilized  eggs  can 
resist  a  much  higher  concentration  of  a  strong  base  than  the 
fertihzed  eggs.  That  this  difference  is  due,  partly  at  least,  to 
the  difference  in  the  rate  of  oxidations  (and  the  developmental 
changes  dependent  upon  these)  is  proved  by  the  fact  that  the 
destructive  action  of  the  alkali  can  be  inhibited  by  the  addi- 
tion of  some  KCN.  Newly  fertilized  eggs  of  purpuratus  were 
distributed  into  the  following  two  solutions : 

(1)  50c.c.m/2  NaCl+l.l  c.c.  m/2  KCl+0.2c.c.  N/10  NaOH 

(2)  50  c.c.  m/2  NaCl+1.1  c.c  m/2  KCl+0.2  c.c.  N/10  NaOH 

+0.5  c.c.  1/10  of  1  per  cent  KCN. 

The  eggs  that  had  been  in  the  first  solution  for  three  and 
one-half  hours  were  all  destroyed  when  they  were  transferred 
to  normal  sea-water.  Those  that  had  been  in  solution  (2)  were 
still  alive  at  that  time  and  could  continue  to  develop  when 
transferred  into  normal  sea-water. 

1  Loeb,  "  Ueber  die  Ursachen  der  Giftigkeit  einer  reinen  Chlornatriumlosung, ' 
Biochem.  Zeitschr.,  II,  81,  1906.  It  is  perhaps  worthy  of  notice  that  the  unferti- 
lized eggs  of  Arbacia  are  more  quickly  injured  by  solutions  of  NaCl  than  are  those 
of  S.  purpuratus. 


Prolongation  of  the  Life  of  the  Egg     87 


In  order  to  indicate  the  greater  resistance  of  the  unferti- 
Hzed  eggs  the  following  experiments  may  he  mentioned.  Un- 
fertilized eggs  of  purpuratus  were  distributed  into  the  following 
two  solutions: 

(1)  50  c.c.  0.54  m  NaCl+1.1  c.c.  in  2  KCl+l.Oc.c.  NVlO  XaOH 

(2)  50  c.c.  0.54  m  NaCl+1 . 1  c.c.  in/2  KCl+1 .0  c.c.  N/10  NaOH  + 

0.5  c.c.  1/10  of  1  per  cent  KCX. 

The  eggs  kept  in  solution  (1)  were  disintegrated  in  about  six 
hours.  The  eggs  of  solution  (2)  were  still  alive  after  forty-one 
hours  and  could  still  be  fertilized  after  they  were  transferred 
into  normal  sea-water.^  (In  such  experiments  the  eggs  should 
not  be  fertilized  immediately  after  they  are  transferred,  l)ut 
several  hours  later.) 

It  could  be  shown  that  lack  of  oxygen  acts  like  the  addi- 
tion of  KCN.2  The  unfertilized  eggs  resist  a  concentration  of 
NaOH  five  times  as  great  as  that  used  for  the  fertilized  eggs. 
This  experiment  also  shows  that  if  the  oxidations  are  retarded 
in  the  egg  by  the  addition  of  KCN  it  is  able  to  resist  the  NaOH 
much  longer. 

But  it  may  be  doul)tful  whether  we  can  account  in  this 
way  for  the  entire  diff(}rence  between  the  sensitiveness  of  ferti- 
lized and  unfertilized  eggs.  We  must  possibly  take  into  con- 
sideration the  fact  that  while  the  fertilized  egg  is  in  direct 
contact  with  the  alkali,  the  unfertilized  egg  is  surrounded  by 
two  shells,  the  chorion  and  the  cortical  layer.  The  cytoplasm 
of  the  unfertilized  egg  is  therefore  in  all  probal)ility  not  exposed 
to  the  same  concentration  of  NaOH  to  which  the  fertilized  egg 
is,  which  has  lost  these  two  layers. 

3.  The  writer  was  able  to  show  generally  that  in  man>' 
abnormal  solutions  in  which  the  unfertilized  oix^  lives  longer 
than  the  fertilized  egg,  the  life  of  the  latter  may  be  prolonged 
by  lack  of  oxygen  or  by  the  suj^i^ression  of  oxidations.     In  a 

1  Loeb,  "  Ueber  die  Hemnmiig  clcr  Ciiftwirkimt;  cli-r  liydro.xylioncii  ;iuf  das 
Seeigelei  mittels  Cyankalium,"  Biochem.  Zcitschr.,  XXVI,  279.  1910. 

2  Loeb,  ibid.,  289. 


88       Artificial  Parthenogenesis  and  Fertilization 

mixture  of  50  c.c.  m/2  NaClH-2.5  c.c.  m/2  CaClg  the  fertilized 
eggs  of  Arbacia  were  injured  after  three  hours  and  ten  minutes 
to  such  an  extent  that  they  were  not  able  to  develop  after  they 
were  transferred  to  sea-water.  All  the  fertilized  eggs  of  the 
same  female  if  kept  the  same  length  of  time  in  the  same  solu- 
tion free  from  oxygen  developed  and  many  reached  the  pluteus 
stage.  Some  of  the  eggs  taken  out  after  seven  hours  from  this 
solution  free  from  oxj^gen  were  still  able  to  develop  into  larvae. 
The  addition  of  five  drops  of  a  1/10  of  1  per  cent  KCN  solution 
to  50  c.c.  of  the  abnormal  solution  acted  just  as  well  if  not 
better  than  lack  of  oxygen.  (This  may  have  been  due  to  the 
fact  that  the  solution  was  slightly  alkaline  which  often  acts 
beneficially  in  solutions  containing  Ca.)  The  same  could  be 
shown  for  analogous  solutions  in  which  the  CaCl2  was  replaced 
by  MgCla,  SrCla,  or  BaCls- 

In  a  mixture  of  49  c.c.  6/8  m  grape  sugar -fl  c.c.  sea-water 
which  was  exposed  to  oxygen  the  eggs  were  injured  to  such  an 
extent  after  three  hours  that  practically  no  egg  was  able  to 
develop  when  transferred  into  sea-water.  Eggs  which  had 
been  for  three  hours  in  the  same  solution  freed  from  oxygen 
were  practically  all  able  to  develop. 

In  this  case  we  were  dealing  with  solutions  of  substances 
for  which  the  egg  is  normally  impermeable.  It  was  of  consider- 
able interest  to  find  out  whether  lack  of  oxygen  has  the  same 
life-saving  effect  if  the  harmful  substance  diffuses  easily  into 
the  egg.  For  this  purpose  distilled  water  and  narcotics  were 
selected.  Newly  fertilized  eggs  of  Arbacia  were  distributed 
into  the  following  solutions: 

(1)  27.5  c.c.  sea-water+22. 5  c.c.  distilled  water 

(2)  27.5  c.c.  sea-water +22. 5  c.c.  distilled  water+5  drops  1/10  of 

1  per  cent  NaCN. 

The  eggs  in  the  first  solution  had  suffered  in  five  hours  and 
forty  minutes  to  such  an  extent  that  no  egg  was  able  to  develop 
into  a  larva,  after  they  were  transferred  to  sea-water.     The 


Prolongation  of  the  Life  of  the  Egg  89 

eggs  in  the  second  solution  when  transferred  at  the  same  time 
to  sea-water  all  developed  into  larvae.  The  fact  that  the  NaCX 
inhibited  the  development  of  the  eggs  in  the  hypotonic  solution 
saved  their  lives.  An  experiment  with  27.5c.c.  sea-water -f- 
22.5  c.c.  6/8  m  ethylalcohol  with  and  without  5  drops  of  1/10 
of  1  per  cent  NaCN  gave  a  similar  result.  In  the  same  way  it 
could  be  shown  that  excessive  amounts  of  chloroform,  chloral 
hydrate,  phenylurethane,  when  applied  in  a  definite  concen- 
tration, injured  the  fertilized  eggs  much  more  rapidly  if  oxygen 
was  present  and  oxidations  were  going  on  in  the  egg,  than  if 
the  oxygen  was  removed  from  the  solution  or  the  oxidations 
were  suppressed  through  the  addition  of  KCN.^ 

4.  The  most  striking  experiments  of  this  kind  are  perhaps 
those  published  by  the  writer  on  the  inhibition  of  the  toxic 
effects  of  hypertonic  solutions  on  the  eggs  of  purpuratus  by 
lack  of  oxygen  or  the  addition  of  KCN,  or  of  chloral  hydrate. 
A  few  examples  may  be  given: 

Eggs  were  fertilized  with  sperm  and  put  eleven  minutes 
later  into  three  flasks,  each  of  which  contained  100  c.c.  of  sea- 
water +16  c.c.  2 J  m  CaCl2.  One  flask  was  in  contact  with  air, 
while  the  other  two  flasks  were  connected  with  a  hydrogen 
generator.  The  air  was  driven  out  from  these  two  flasks  before 
the  beginning  of  the  experiment.  The  eggs  were  transferred 
from  one  of  these  flasks  after  four  hours  and  fourteen  minutes, 
from  the  second  flask  after  five  hours  and  twenty-nine  minutes, 
into  normal  (aerated)  sea-water.  The  eggs  that  had  been  in 
the  hypertonic  sea-water  exposed  to  air  were  transferred  simul- 
taneously with  the  others  into  separate  dishes  with  aerated 
normal  sea-water.  The  result  was  most  striking.  Those  eggs 
that  had  been  in  the  hypertonic  sea-water  with  air  were  all 
completely  disintegrated  in  a  way  which  I  will,  for  the  sake  of 
briefness,  designate  as  ''black  cytolysis"   (Figs.  36,  37,  38). 

1  Loeb,  "Die  Hemmung  verschiedener  Giftwirkunson  auf  das  bL'fruchtote 
Seeigelei  durch  Hemmung  der  Oxydationen  in  demsellji-n,  "  Biochem.  Zeitschr., 
XXIX.  80,  1910.  It  might  be  well  to  indicate  that  these  experiments  also  contra- 
dict the  idea  that  narcosis  is  due  to  asphyxiation. 


Fig.  32 


Fig.  33 


Fig.  34 


O    *. 


^^-O'.o'' 


•O     o  o.  o°^^-Qo'o©.»  o»^^ 


ft  o 


.o?oM°£&^°' 


O   'Jo 

Q-O 


?D;- 


0 


0^0 
0"o 


O  V,:^-' 

0  o        C 


o 


<3  "    O 

o» 


o'ot'ao  o„°a© 


o  ^  - 


°  0 

0 


C 


Fig.  36 


Fig.  37 


Fig.  38 

Figs.  32-37. — ^Typical  disintegration  of  a  sea-urchin  egg,  which  had  been 
treated  for  some  time  with  aerated  hypertonic  sea-water. 

Fig.  38. — Another  type  of  "black  cytolysis." 


Prolongation  of  the  Life  of  the  Egg  91 


Ten  per  cent  of  the  eggs  had  been  transformed  into  ''shadows" 
(white  cytolysis).  It  goes  without  saying  that  all  the  eggs  that 
had  been  in  the  aerated  hypertonic  sea-water  five  and  a  half 
hours  were  also  dead.  The  eggs  that  had  been  in  the  same 
solution  in  the  absence  of  oxygen  appeared  all  normal  when 
they  were  taken  out  of  the  solution,  and  three  hours  later — 
the  temperature  was  only  15°  C. — they  were  all,  without  excep- 
tion, in  a  perfectly  normal  two-  or  four-cell  stage.  The  further 
development  was  also  in  most  cases  normal.  They  swam  as 
larvae  at  the  surface  of  the  vessel  and  went  on  the  third  day 
(at  the  right  time)  into  a  perfectly  normal  pluteus  stage,  after 
which  their  observation  was  discontinued.  Of  the  eggs  that 
had  been  five  and  a  half  hours  in  the  hypertonic  sea-water 
deprived  of  oxygen,  about  90  per  cent  segmented.^ 

In  another  experiment  newly  fertilized  eggs  of  purpuratus 
were  put  into  two  dishes  each  containing  50  c.c.  sea-water -}- 
15  c.c.  2J  m  NaCl.  To  the  one  dish  was  added  1  c.c.  1/10  of 
1  per  cent  KCN.  The  eggs  remained  305  minutes  in  the  solu- 
tion. When  they  were  put  back  into  normal  sea-water  many  of 
those  that  had  been  in  the  dish  containing  KCN  developed  per- 
fectly normally  into  plutei,  while  those  that  had  been  in  the 
solution  without  KCN  all  disintegrated  into  droplets  in  about 
one-half  hour.  These  experiments  were  often  repeated  and 
they  are  indeed  very  striking  demonstration  experiments.  They 
show  that  the  inhibition  of  oxidations  in  these  experiments 
protects  the  egg  against  the  injurious  effects  of  the  hypertonic 
solution  for  a  considerable  time. 

Chloral  hydrate  had  also  a  very  slight  protective  action. 
When  2.5  c.c.  N/10  chloral  hydrate  were  added  to  68  c.c.  of  the 
hypertonic  solution,  about  5  per  cent  of  the  eggs  were  saved 
under  conditions  under  which  KCN  saved  all  the  eggs.^ 

1  Loeb,  University  of  California  Publications,  Physiology,  III,  49,  1906. 

2  Loeb,  "Ueber  die  Hemmung  der  toxischen  Wirkung  hypertonischer  Losun- 
gen  auf  das  Seeigelei  durch  Sauerstoffmangel  und  Cyankalium,"  FjlUger's  Archiv, 
CXIII,  487.   1906. 


92      Artificial  Parthenogenesis  and  Fertilization 


It  is  very  characteristic  that  the  eggs  usually  disintegrate 
not  while  they  are  in  the  hypertonic  solution  but  after  they 
have  been  put  back  into  the  normal  sea-water.  This  agrees  with 
the  observation  we  made  on  the  disintegration  of  the  egg  after 
membrane  formation,  namely,  that  this  disintegration  begins 
at  the  time  of  the  first  cell  division.  The  hypertonic  solution 
does  not  permit  cell  division,  while  the  cell  division  begins  in  an 
irregular  way  after  the  eggs  are  transferred  to  normal  sea-water. 
Such  eggs  disintegrate  into  little  droplets. 

The  unfertilized  eggs  do  not  suffer  as  quickly  in  the  hyper- 
tonic solution  as  the  fertilized  eggs.  In  the  unfertilized  egg 
the  hypertonic  solution  must  first  induce  some  parthenogenetic 
changes  before  it  can  produce  its  destructive  action. 

5.  We  must  in  this  connection  consider  an  idea  emphasized 
by  various  authors  that  the  fertilized  egg  is  more  permeable 
than  the  unfertilized.  On  this  assumption  we  might  understand 
why  the  fertilized  egg  is  more  easily  destroyed  in  abnormal  salt 
solutions  than  the  unfertilized  egg.  Leaving  aside  temporarily 
the  fact  that  the  fertilized  egg  is  protected  for  some  time  against 
the  action  of  the  same  agencies  if  its  development  is  prevented 
by  lack  of  oxygen,  this  idea  meets  with  other  difficulties.  If 
this  view  were  correct,  the  egg  should  be  permeable  for  such 
substances  as  NaOH,  and  the  fertilized  egg  much  more  so  than 
the  unfertilized  egg.  The  observations  of  Warburg  and  Harvey 
to  be  mentioned  later  have  definitely  shown  that  this  is  not  the 
case.  But  how  are  we  then  to  explain  such  phenomena  as  the 
following?  The  egg  of  Arbacia  possesses  a  red  pigment.  If 
fertilized  and  unfertilized  eggs  are  put  into  an  alkaline  solution, 
e.g.,  50c.c.  m/2  NaCl-fl  c.c.  m/2  BaCl2+0.2  c.c.  N/10 
NaOH,  the  fertilized  eggs  lose  their  pigment  almost  instantly 
while  the  unfertilized  eggs  keep  it  for  a  long  time..^  The  explana- 
tion lies  probably  in  the  fact  that  the  alkaline  solution  comes 
into  direct  contact  with  the  cytoplasm  of  the  fertilized  egg,. 

1  Loeb,  Biochem.  Zeitschr.,  XXIX,  93,  1910. 


Prolongation  of  the  Life  of  the  Egg  93 

while  the  cytoplasm  of  the  unfertilized  e^g  is  surrounded  by  a 
chorion  and  by  the  cortical  lajTr,  which  ])rotect  it.  Hence  the 
surface  layer  of  the  fertilized  egg  is  more  rapidly  injured  than 
that  of  the  unfertilized  egg,  and  this  may  account  for  the  more 
rapid  escape  of  pigment  from  the  former. 

The  fact  that  KCN  or  lack  of  oxygen  prolongs  the  life  of 
the  egg  in  abnormal  solutions  might  be  interpreted  as  indicat- 
ing that  the  KCN  prevents  the  diffusion  of  abnormal  solutions 
into  the  egg.  Wasteneys  and  the  writer  investigated  this  possi- 
bility in  the  fertilized  eggs  of  purpuratus  which  were  stained 
red  with  neutral  red.  Such  eggs  are  rapidly  destroyed  in  a 
mixture  of  NaCl+KCl  to  which  some  N/10  NH4OH  is  added; 
while  the  addition  of  KCN  prolongs  their  life  in  such  a  solution. 
This  action  was  not  caused  by  a  retardation  of  the  diffusion  of 
NH4OH  into  the  egg  through  the  presence  of  KCN,  for  it  was 
found  that  the  eggs  were  just  as  quickly  stained  yellow  in  the 
solutions  of  NH4OH  containing  KCN  as  in  those  free  from  it. 
The  life-saving  action  of  KCN  is  therefore  not  due  to  any  effect 
upon  the  permeability  of  the  egg. 

All  the  facts  agree  with  the  assumption  that  the  egg  is  more 
sensitive  to  abnormal  solutions  when  its  rate  of  oxidations  is 
high  or  when  certain  developmental  changes,  e.g.,  cell  divisions, 
take  place  than  when  it  is  at  rest. 

There  is,  however,  an  apparent  exception  to  the  rule  that 
the  fertilized  egg  is  more  easily  injured  than  the  unfertilized; 
weak  bases  like  NH4OH  or  the  amines  injure  unfertilized  eggs 
more  rapidly  than  fertilized  eggs.  When  we  put  unfertilized 
eggs  of  Arhacia  for  twenty  minutes  at  23°  C.  into  a  mixture  of 
50  c.c.  m/2  NaCl+KCl+CaClo-hO.3  c.c.  N/10  NH4OH  and 
then  transfer  them  to  sea-water,  they  will  begin  to  segment 
but  will  disintegrate  very  rapidly.  If  we  put  newly  fertilized 
eggs  for  twenty  minutes  into  the  same  solution  we  find  that 
the  short  stay  in  the  ammonia  solution  does  not  hurt  them.  In 
this  experiment  the  injury  to  the  unfertilized  eggs  is  oul\-  the 


94       Artificial  Parthenogenesis  and  Fertilization 

indirect  effect  of  the  ammonia  solution.  The  latter  induces  the 
development  in  the  unfertilized  eggs,  but  if  they  are  not 
treated  with  the  hypertonic  solution  they  will  disintegrate. 

The  case  is  similar  to  our  experience  with  artificial  mem- 
brane formation  by  weak  acids.  The  eggs  will  die  after  this 
treatment  if  they  do  not  receive  the  necessary  second  treat- 
ment. It  is  not  the  weak  acid  which  kills  the  eggs  in  this 
case  but  the  membrane  formation  without  the  addition  of  the 
second  factor.  If  fertilized  eggs  receive  the  same  treatment 
with  a  weak  acid  they  will  not  suffer  at  all. 


XI 

FURTHER  EXPERIMENTS  OX  THE  ACTION  OF  THE 
HYPERTOXIC  SOLUTION  AFTER  MEMBRANE 

FORMATION 

1.  The  most  convenient  and  reliable  method  of  saving  the 
eggs  of  the  sea-urchin  from  disintegrating  after  artificial  mem- 
brane formation  consists  in  subjecting  them  to  a  short  exposure 
to  hypertonic  sea-water.  If  they  are  then  transferred  at  the 
right  time  from  this  hypertonic  solution  to  normal  sea-water, 
they  all  segment  and  develop  into  larvae;  and  in  a  certain  per- 
centage of  these  larvae  the  development  is  perfectly  normal. 

Why  do  not  all  the  eggs  so  treated  produce  normal  larvae  ? 
In  order  to  understand  this  we  must  consider  two  facts.  If  the 
eggs  are  left  too  short  a  time  in  the  hypertonic  solution,  the 
threatening  disintegration  is  not  prevented  and  the  eggs  go  to 
pieces  precisely  as  though  no  hypertonic  solution  had  been 
used.  If  the  eggs  are  left  too  long  in  the  solution  they  do 
indeed  all  develop  into  larvae,  but  the  larvae  are  abnormal; 
the  farther  the  proper  moment  for  transference  of  the  eggs  to 
normal  sea-water  has  been  overshot,  the  more  abnormal  the 
larvae  are  and  the  earlier  they  die. 

Now  the  proper  length  of  exposure  to  the  hypertonic  solu- 
tion is  not  the  same  for  all  eggs.  Even  if  the  eggs  of  the  same 
female  are  used,  exposed  for  the  same  length  of  time  to  the 
fatty  acid  and  placed  for  the  same  time  in  the  same  hypertonic 
solution,  it  will  be  found  that  the  correct  time  of  exposure  varies 
within  fairly  wide  limits  for  different  eggs.  This  can  easily  be 
seen  if  the  eggs  are  brought  back  from  the  hypertonic  solution 
into  normal  sea-water  at  intervals  of  five  minutes.  Perhaps 
none  of  the  eggs  first  returned  to  normal  sea-water  develo}). 
until  one  reaches  a  portion  in  which  all  grow  into  larvae;    but 

95 


96       Artificial  Parthenogenesis  and  Fertilization 

not  all  form  normal  larvae.  Some  of  the  eggvs  have  remained 
too  long  in  the  hypertonic  solution  and  this  over-exposure 
injures  the  eggs  concerned.  This  is  proved  by  the  fact  that  the 
number  of  injured  larvae  is  always  the  greater  the  later  the  eggs 
were  taken  out  of  the  hypertonic  solution.  What  determines 
this  individual  variation  we  camiot  say.  In  part  it  is  connected 
with  the  unequal  distribution  of  the  eggs  in  the  vessel,  whereby 
the  access  of  oxj^gen  to  some  eggs  is  more  complete  than  to 
others.  This  is,  however,  only  one  of  several  factors.  It  is  an 
important  fact  that  even  a  slight  over-exposure  to  the  hyper- 
tonic solution  injures  the  eggs. 

How  long  ought  the  eggs  to  remain  in  the  hypertonic  solu- 
tion? This  again  depends  upon  how  long  after  membrane 
formation  they  are  placed  in  the  hypertonic  solution.  A.  R. 
Moore  has  carried  out  some  experiments  on  this  subject  in  my 
laboratory  on  S.  purpuratus.  He  determined  the  time  which  is 
necessary  to  cause  all  the  eggs  to  develop  into  larvae.  When 
the  eggs  are  placed  in  the  hypertonic  sea-water  (50  c.c.  of 
sea-water +8  c.c.  2|  m  NaCl)  immediately,  i.e.,  two  to  four 
minutes  after  membrane  formation,  they  must  remain  sixty 
to  seventy  minutes  in  the  hj^pertonic  solution  before  all  can 
be  caused  to  develop.  When  placed  in  the  hypertonic  solution 
thirty  minutes  after  membrane  formation,  they  must  remain  in 
it  forty  to  fifty  minutes  in  order  to  obtain  the  best  results.  If 
transferred  to  the  hypertonic  solution  one  or  two  hours  after 
artificial  membrane  formation,  it  was  only  necessary  to  leave 
them  in  it  thirty  to  forty  minutes  in  order  to  get  all  the  eggs  to 
develop  into  larvae.  If  a  still  longer  interval  elapses  before  the 
eggs  are  put  in  the  hypertonic  sea-water  after  membrane  forma- 
tion, the  results  again  become  worse.  In  these  experiments  the 
temperature  was  about  12°  C. 

In  order  to  give  the  reader  a  more  perfect  idea  of  what  has 
been  said  I  will  describe  more  completely  one  of  my  own  obser- 
vations.    Membrane  formation  was  produced  in  the  eggs  by 


Action  of  the  Hypertonic  Solution 


97 


treating  them  for  two  minutes  with  50  c.c.  of  sea-water4-2 . 9  e.c. 
N/10  butyric  acid  (which  was  thoroughly  mixed).  One  portion 
of  the  eggs  (A)  was  placed  at  once  in  hypertonic  sea-water 
(50  c.c.  of  sea-water +8  c.c.  2 J  m  NaCl),  a  second  portion  (B) 
thirty  minutes  later,  and  a  third  portion  (C)  after  two  hours. 
At  intervals  of  five  minutes  a  portion  of  the  eggs  was  replaced 
in  normal  sea-water,  the  number  of  eggs  undergoing  segmenta- 
tion ascertained,  and,  on  the  following  day,  the  number  of 
larvae  that  had  been  produced  was  counted.  Table  Yl  gives 
the  result.  The  upper  horizontal  line  shows  how  long  (in 
minutes)  the  eggs  remained  in  the  hypertonic  solution  and 
underneath  is  shown  the  percentage  of  the  eggs  in  each  part 
that  grew  into  larvae. 

TABLE  VI 

Percextage  of  the  Eggs  Which  Developed  into  Larvae  after 

Remaining  in  the  Solution 


5' 

10' 

15' 

20' 

25' 

30' 

35' 

40' 

45' 

50' 

A 

0 

0 

0 

0 

0 

1 

30 

90 

99 

100 

B 

0 

0 

1 

2 

40 

70 

90 

98 

C 

0 

i 

10 

30 

70 

90 

100 

The  temperature  of  the  hj'pertonic  solution  was  15°  C. 

What  is  the  cause  of  this  variation  in  time  required  for  the 
exposure  to  the  hypertonic  solution?  I  suspect  that  it  has 
something  to  do  with  changes  which  occur  in  the  egg  after  the 
membrane  formation.  One  of  these  changes  is  the  formation 
of  a  fine  gelatinous  layer  around  the  cytoplasm,  which  does  not 
begin  until  at  least  ten  or  fifteen  minutes  after  the  membrane 
formation.  It  may  be  that  the  hypertonic  solution  only  or 
mainl}^  takes  effect  after  a  definite  change  has  taken  place  in 
the  egg. 

In  all  the  experiments  hereafter  mentioned,  the  eggs  were 
transferred  some  ten  minutes  after  membrane  formation;  hence 
the  length  of  exposure  is  somewhat  as  in  series  A. 


98       Artificial  Parthenogenesis  and  Fertilization 


2.  We  will  now  inquire  how  the  time  which  the  eggs  must 
remain  in  the  hypertonic  solution  varies  with  the  osmotic  pres- 
sure of  the  latter.^ 

The  eggs  of  a  sea-urchin  were  treated  with  sea-water  con- 
taining butyric  acid,  and  all  formed  membranes.  The  eggs 
were  then  distributed  among  solutions  consisting  of  50  c.c.  of 
sea-water +respectively,  0,  1,  2,  3,  4,  5,  6,  7,  8,  10,  12,  and 
14  c.c.  of  2i  m  (grammolecular)  NaCl  solution.  A  portion  of 
the  eggs  was  transferred  from  each  of  these  solutions  to  normal 
sea-water  after  33,  45,  57,  68,  98,  and  128  minutes,  and  the 
percentage  that  developed  into  larvae  determined  for  each 
portion.  Table  VII  gives  the  result.  The  temperature  of  the 
hypertonic  solution  was  16°  C. 


TABLE  VII 


Naturk  of  the  Solution 

Percentage  of  the  Eggs  of 

S.  purpuratus  Which  Developed 

into  Larvae  after  Remaining  in 

the  Solution 

33 
min. 

45 
min. 

57 
min. 

68 
min. 

98 
min. 

128 
min. 

50  c.c.  of  sea-water 

0 
0 
0 
0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
1 
1 
0 
0 
0 

0 

0 

0 

2 

30 

50 

10 

a  few 

0 

0 

0 

1 

50 

70 

80 

10 

0 

0 

0 

0 

30 

100 

70 

a  few 

1 

0 

0 

0 
a  few 
60 
10 

0 

0 

0 

0 

0 

50  c.c,  of  sea-water  +  4  c.c.  2^  m  NaCl 
50  c.c.  of  sea-water -i-  5  c.c.  2|  m  NaCl 
50  c.c.  of  sea-water  +  6  c.c.  2^  m  NaCl 
50  c.c.  of  sea-water -I-  7  c.c.  2^  m  NaCl 
50  c.c.  of  sea-water  +  8  c.c.  2^  m  NaCl 
50  c.c.  of  sea-water +10  c.c.  2^  m  NaCl 
50  c.c.  of  sea-water 4- 12  c.c.  2|  m  NaCl 
50  c.c.  of  sea-water -1-14  c.c.  2^  m  NaCl 

This  brings  out  two  facts.  In  the  first  place  the  effective- 
ness of  the  h>T)ertonic  solution  has  definite  limits.  The  addi- 
tion of  4  c.c.  of  2i  m  NaCl  is  too  small,  the  addition  of  12  c.c.  too 
much,  and  in  the  latter  case  the  eggs  go  to  pieces  by  disinte- 
grating into  drops.  Secondly,  we  see  that  when  once  the  opti- 
mum concentration  is  reached,  i.e.,  that  which  produces  the 

1  Loeb,  "Ueber  den  Unterschied  zwischen  isosmotischen  und  isotonischen 
Losungen  bei  der  kiinstUchen  Parthenogenese,"  Biochem.  Zeitschr.,  XI,  144,  1908. 


Action  of  the  Hypertonic  Solution  99 


highest  percentage  of  larvae  (viz.,  7  c.c.  of  2|  m  NaCl),  a  further 
increase  in  concentration  does  not  decrease  the  time  during 
which  the  egg  has  to  remain  in  the  hypertonic  solution.  This 
has  some  bearing  on  the  theory  of  the  action  of  the  hypertonic 
solution.  Since  the  loss  of  water  by  the  egg  in  this  solution 
increases  with  its  concentration,  it  is  obvious  that  the  effect  of 
the  hypertonic  solution  does  not  grow  in  proportion  with  the 
loss  of  water  on  the  part  of  the  egg. 

This  experiment  has  been  very  often  repeated  with  an  essen- 
tially similar  result.  The  addition  of  only  3  c.c.  of  2|  m  NaCl 
to  50  c.c.  of  sea-water  has  never  led  to  the  development  of  a 
larva  of  S.  purpuratus  after  membrane  formation,  however 
long  the  eggs  are  left  in  the  solution. 

An  example  of  such  an  experiment  with  a  longer  range  of 
time  should  be  mentioned.  The  unfertilized  eggs  of  a  female 
S.  purpuratus  were  placed  after  artificial  membrane  formation  in 
50  c.c.  of  sea-water +0,  1,  2,  3,  4,  etc.,  of  2 J  m  NaCl.  After  37, 
47,  57, 110, 150,  200,  280,  and  340  minutes  a  portion  of  eggs  from 
each  bowl  was  replaced  into  normal  sea-water.  Temperature 
16.5°  to  17.5°  C. 

This  experiment  described  by  Table  VIII  again  demon- 
strates that  the  addition  of  less  than  4  c.c.  of  2§  m  NaCl  to 
50  c.c.  of  sea-water  is  not  sufficient  to  protect  the  eggs  from 
decay  and  evoke  development  even  after  prolonged  exposure. 
Moreover,  it  is  clear  that  when  once  the  optimum  is  reached 
the  time  of  exposure  cannot  be  decreased  by  a  further  raising 
of  the  concentration  of  the  sea-water.  When  8  c.c.  of  2J  m 
NaCl  is  added,  the  minimum  length  of  exposure  is  47  minutes, 
and  the  same  is  the  case  when  12  c.c.  of  2J  m  NaCl  is  added. ^ 

Perhaps  the  following  facts  explain  why  it  is  not  possible  to 
save  eggs  from  disintegrating  by  the  addition  of  less  than  4  c.c. 
of  2J  m  NaCi  to  50  c.c.  of  sea-water  after  artificial  membrane 

iJn  these  experiments  the  eggs  were  placed  in  the  hypertonic  solution  very 
soon  after  membrane  formation;  otherwise  the  times  would  have  been  shorter. 


100     Artificial  Parthenogenesis  and  Fertilization 


formation.  If  eggs  fertilized  with  sperm  are  placed  in  50  e.c. 
of  sea-water,  to  which  increasing  amounts  of  2i  m  NaCl  are 
added,  it  can  be  seen  that  the  addition  of  1  c.c.  of  2i  m  NaCl 
to  50  c.c.  of  sea-water  has  no  effect  on  the  development;  the 
addition  of  2  c.c.  of  2J  m  NaCl  retards  the  first  division,  but  to  a 
scarcely  noticeable  degree;  while  the  addition  of  3  c.c.  increases 

TABLE  VIII 


Nature  of  the  Solution 


Percentage  of  Eggs  That  Developed  intoLarv.ve 
AFTER  Remaining  in  the  Solution 


37 
min. 


47 
min. 


57 
min. 


50    C.C    of    sea-water+2  c.c. 

2h  m  NaCl 

50    c.c.    of    sea-water+3    c.c. 

2^  m  XaCl 

50    c.c.    of    sea-water +4    c.c. 

2^  m  NaCl 

50    c.c.    of    sea-water+5    c.c. 

2h  m  NaCl 

50    c.c.    of    sea-water -|-6    c.c. 

2|  m  NaCl 

50    c.c.    of    sea-water+8    c.c. 

2|  m  NaCl 

50   c.c.   of   sea-water +  10   c.c. 

2|  m  NaCl 

50  c.c.   of  sea-water+12   c.c. 

2|  m  NaCl 

50   c.c.   of   sea-water+14   c.c. 

2|  m  NaCl 


0 
0 
0 
0 

0 
a  few 
0 
0 


0 
0 
0 
0 

10 

8 
1 
0 


0 

0 

0 

30 

80 

100 

30 

2 

0 


110 
min. 


150 
min. 


200 
min. 


0 

0 

1 

50 

95 


0 

0 

5 

80 


0 
0 

8 
90 


0 

a  few 


0 
0 


280 
min. 


0 
0 

90 


0 
0 


340 
min. 


0 
0 

30 
40 


0 
0 


0 
0 


the  time  of  the  first  division  by  about  12  per  cent  of  its  usual 
length,  although  the  development  of  the  eggs  proceeds  ahnost 
normally.  The  addition  of  4  c.c.  of  2|  m  NaCl  to  50  c.c.  of 
sea-water  is  the  first  to  delay  the  first  cleavage  for  several  hours 
(over  100  per  cent  of  the  normal  length) ;  it  is  true  that  the  eggs 
can  develop  very  slowly  in  this  solution,  but  not  all  the  eggs  are 
able  to  develop.  Hence  it  appears  that  here  too  the  addition 
of  4  c.c.  of  NaCl  is  of  critical  importance.  In  50  c.c.  of  sea- 
water+5  c.c.  2^  m  NaCl  the  eggs  do  not  develop  beyond  the 
thirty-two-  to  sixty-four-cell  stage.     Probably,  therefore,  in  a 


Action  of  the  Hypertonic  Solution  101 


solution  of  50  c.c.  of  sea-water +4  c.c.  of  2 J  m  NaCl  considerable 
changes  take  place  in  the  chemical  reactions  within  the  egg. 
These  changes  lead  to  those  effects  which,  after  membrane 
formation,  allow  the  egg  to  develop  normally  upon  transfer- 
ence to  ordinary  sea-water. 

3.  The  third  factor  which  determines  the  length  of  exposure 
to  the  hypertonic  solution  is  the  temperature.  In  my  first 
papers  upon  artificial  parthenogenesis  I  was  undecided  as  to 
whether  the  hypertonic  solution  has  a  purely  physical  or  a  purely 
chemical  action.  I  had  found  in  1892  that  it  prevents  cell  divi- 
sion more  quickly  than  nuclear  division.  Since  a  hypertonic 
solution  prevents  cell  division  more  easily  than  nuclear  division, 
one  can  obtain,  at  a  certain  minimal  grade  of  the  hj^pertonicity 
of  the  solution,  nuclear  division  without  cell  division.  It  is 
quite  possible  (though  not  proven)  that  this  prevention  of 
cleavage  depends  upon  the  rise  in  viscosity  of  the  protoplasm, 
owing  to  the  withdrawal  of  water  from  it  in  the  hypertonic  solu- 
tion. On  the  other  hand,  it  was  not  very  probable  that  the 
activation  of  the  unfertilized  egg  by  a  hypertonic  solution  could 
be  referred  to  a  physical  effect. 

The  determination  of  the  temperature  coefficient  affords  us 
a  ready  means  of  differentiating  whether  a  given  physiological 
process  depends  upon  a  chemical  reaction  or  upon  a  purely 
physical  change.  As  van't  Hoff  and  Arrhenius  have  shown, 
the  temperature  coefficient  for  chemical  reactions  is  relatively 
high,  viz.,  not  less  than  2  for  10°  C,  while  physical  processes 
possess  in  general  a  lower  temperature  coefficient.  We  can  in 
this  way  set  up  a  criterion  whether  the  effect  of  the  hypertonic 
solution  upon  the  egg  after  artificial  membrane  formation 
depends  upon  the  influence  of  a  chemical  reaction  within  the 
egg  or  a  physical  process.^ 

1  In  my  first  researches  upon  the  eflfects  of  salts.  I  was  troubled  because  I 
possessed  no  criterion  to  decide  whether  I  had  to  deal  with  purely  physical  phe- 
nomena    e.g..    coagulations,    or   with   chemical    processes.     Cohen's    admirable 
Vorlesungen  ueber  physicalische  Chemie  fur  Arzte  indicated  the  importance  of  the 


102     Artificial  Parthenogenesis  and  Fertilization 

AYith  this  object  in  view,  the  following  experiments  were 
performed.  Mem])rane  formation  was  produced  by  treating 
eggs  of  S.  purpuratus  with  butyric  acid.  The  eggs  were  then 
divided  into  two  dishes  containing  the  same  hypertonic  solution 
(50  c.c.  of  sea-water +8  c.c.  2 J  m  NaCl).  One  of  these  cUshes 
was  maintained  at  a  certain  temperature,  the  other  at  a  tem- 
perature 10°  higher.  At  different  intervals,  portions  of  the  eggs 
were  replaced  in  normal  sea-water  of  room  temperature,  and  the 
minimum  exposure  necessary  to  allow  a  certain  percentage  of  the 
eggs  to  develop  was  noted.  The  results  for  the  eggs  of  five 
different  sea-urchins  (S.  purpuratus)  are  contained  in  Table 
IX.  One  noteworthy  result  was  that  at  temperatures  above 
20°  C.  the  eggs  were  harmed  by  the  hypertonic  solution.  Of 
course,  this  does  not  hold  for  the  eggs  of  all  sea-urchins,  as  my 
experiments  at  Woods  Hole  were  usually  performed  at  a  tem- 
perature of  over  20°  C.  It  is  probably  connected  with  the  fact 
that  the  temperature  of  the  water  at  Pacific  Grove  never  is  as 
high  as  that  at  Woods  Hole  in  summer. 

TABLE  IX 


Temperature 

Minimum  Exposiu-e 
for  Production  of 
Numerous  Larvae 

Temperatiu-e  Coefficient 
Qio  for  10°C. 

T.        1    \  4°-5°  C 

210  minutes 
40  minutes 

160  minutes 
about    55  minutes 
about  112  minutes 
about    32  minutes 
about    50  minutes 
all  eggs  die 
about    35  minutes 
all  eggs  die 

(                Qio  =  5 

E^Pl)l5°C 

T^               r\      \        O      V-/  ■••••>••....• 

1           Qxo-3 

}                 Qio  =  3  .  5 

E^p-2)i.5°c : : 

,;,                  ^       U0°    C 

i^xp.  ^••^20°C 

Exp.  4.  jl^Ig;;;  ;;;;;;;;;• 

E-p-^-i26°c::::::::::::: 

temperature  coefficient  in  settling  this  question.  I  myself  applied  this  criterion  to 
the  sphere  of  the  physiology  of  development,  and  caused  my  pupils,  C.  D.  Snyder 
and  S.  S.  Maxwell,  to  employ  it  in  settling  the  question  whether  the  heart  beat 
and  the  transmission  of  nervous  impulses  depend  upon  chemical  or  physical  pro- 
cesses. The  temperature  coefficient  found  in  the  heart  beat  was  of  the  order  of 
magnitude  for  chemical  processes. 


Action  of  the  Hypertonic  Solution  103 

It  is  clear  that  the  temperature  coefficient  Qio  is  higher  than 
2,  in  the  neighborhood  of  3,  and  that  it  quickly  increases  at 
temperatures  which  are  close  to  0°,  and  reaches  the  value  5  or 
perhaps  still  higher  values.^  Hence  the  order  of  magnitude  of 
the  temperature  coefficient  indicates  that  we  are  dealing  here 
with  a  chemical  effect  of  the  hypertonic  solution. 

4.  We  are  in  a  position  to  determine  more  closely  the  nature 
of  the  chemical  processes  upon  which  the  efficiency  of  the  hyper- 
tonic solution  depends,  namely  oxidations.  The  efficacy  of 
the  hypertonic  solution  is  completely  removed  if  we  inhibit 
oxidations  in  the  egg  by  means  of  potassium  cyanide  or  by  the 
withdrawal  of  oxygen.^ 

It  can  be  shown  that  the  treatment  of  eggs  after  artificial 
membrane  formation  with  hypertonic  sea-water  remains  in- 
effective if  the  latter  contains  an  insufficient  amount  of  oxygen, 
or  if  it  contains  KCN.  After  removal  from  such  solutions, 
the  eggs  behave  as  if  only  membrane  formation  had  been 
produced,  i.e.,  they  begin  to  develop,  but  quickly  disintegrate 
in  the  manner  described  in  the  previous  chapter.  A  single 
example  will  suffice  to  illustrate  this. 

Unfertilized  eggs  of  S.  purpuratus  were  placed  for  one  and 
one-half  to  two  minutes  in  50  c.c.  of  sea-water +3  c.c.  N/10 
butyric  acid,  and,  as  usual,  all  formed  a  perfect  fertilization 
membrane  upon  transference  to  sea-water.  They  were  then 
distributed  over  the  following  solutions: 

(1)  50  c.c.  of  sea-water+8  c.c.  of  2^  m  NaCl 

(2)  50  c.c.  of  sea-water+8  c.c.  of  2^  m  NaCl+2  c.c.  1/20  of  1  per 
cent  KCN. 

After  30,  40,  50,  135,  195,  285,  335,  385,  450,  and  1,320  minutes, 
a  sample  of  the  eggs  was  transferred  to  normal  sea-water.  The 
temperature  was  18°  C.     For  the  eggs  in  the  first  solution,  the 

1  Loeb,  U ntersuchungen,  p.  494;  University  of  California  Publications,  Physiol- 
ogy, III,  39,  1906. 

2  Loeb,  Biochem.  Zeitschr.,  I,  194,  1906. 


104    Artificial  Parthenogenesis  and  Fertilization 


result  was  as  follows.  Thirty  minutes  in  the  hypertonic  solu- 
tion was  not  enough,  and  all  the  eggs  disintegrated  except  a 
few  which  developed.  Forty  and  fifty  minutes  were  sufficient 
and  about  50  per  cent  of  the  eggs  developed  into  larvae.  The 
eggs  that  had  been  135  minutes  and  longer  in  the  hypertonic  solu- 
tion went  to  pieces.  None  of  the  eggs,  on  the  other  hand,  whicli 
had  been  in  the  hypertonic  solution  with  potassium  cyanide  (in 
which  the  oxidations  were  inhibited)  developed,  whose  exposure 
to  that  solution  had  been  less  than  385  minutes.  All  such 
eggs  disintegrated  in  the  course  of  the  next  twenty-four  hours, 
and  in  the  same  manner  as  eggs  which  are  left  in  normal 
sea-water,  not  transferred  to  hypertonic  solutions  after  mem- 
brane formation.  Hence  the  effect  of  the  hypertonic  solution 
was  nullified  by  the  addition  of  potassium  cyanide.  A  small 
number — some  two  to  four  in  a  watch  glass  containing  many 
thousands  of  eggs — of  the  eggs,  which  were  in  the  hypertonic 
solution  for  more  than  385  minutes,  developed  into  blastulae. 
Again,  the  eggs  which  had  been  longest  in  the  hypertonic  sea- 
water  with  potassium  cyanide  did  not  go  to  pieces  so  rapidly 
when  transferred  to  normal  sea-water,  as  those  whose  exposure 
thereto  had  been  of  shorter  duration.  This  probably  is  con- 
nected with  the  fact  that  eggs  which  remain  longer  in  the 
cyanide  sea-water  take  up  more  KCN  or  HCN.  This  experi- 
ment was  repeated  very  often,  and  always  with  the  same  result. 
Usually  the  addition  of  a  sufficient  quantity  of  potassium 
cyanide  to  the  hypertonic  sea-water  completely  inhibited  the 
action  of  the  latter.  It  is  worthy  of  mention  that  the  KCN 
does  not  prevent  but  only  retards  the  oxidations,  and  this 
retardation  is  less  the  less  KCN  is  added. 

It  might  be  objected  that  in  the  previous  experiment  potas- 
sium cyanide  had  killed  the  eggs,  or,  at  all  events,  rendered  them 
incapable  of  development.  The  following  experiment,  however, 
will  overthrow  this  idea. 

After  artificial  membrane  formation  (by  means  of  butyric 


Action  of  the  Hypertonic  Solution 


105 


acid)  the  eggs  of  a  female  were  divided  between  two  dishes, 
each  containing  50  c.c.  of  sea-water+8  c.c.  of  2^  m  NaCl.  To 
one  dish  2  c.c.  of  1/20  of  1  per  cent  KCN  were  added.  Samples 
of  the  eggs  were  transferred  at  intervals  of  ten  minutes  from  the 
hypertonic  to  normal  sea-water. 

We  will  consider  first  the  behavior  of  the  eggs  which  had 
been  in  the  hypertonic  solution  without  potassium  cyanide. 
None  of  the  eggs  transferred  from  the  hypertonic  to  normal 
sea-water  before  thirty-five  minutes  had  elapsed  developed; 
some  5  per  cent  of  those  transferred  to  normal  sea-water  after 
thirty-five  minutes  developed  into  good  larvae;  practically 
all  of  those  transferred  after  forty-five  minutes  developed,  and 
the  majority  to  normal  larvae.  Of  those  transferred  after 
fifty-five  minutes  practically  all  developed,  but  only  20  per  cent 
gave  rise  to  normal  larvae,  while  with  eggs  left  still  longer  in 
the  hypertonic  solution  the  results  became  worse  with  increasing 
length  of  exposure. 

Not  a  single  one  of  the  eggs  which  had  been  in  the  hyper- 
tonic sea-water  with  potassium  cyanide  for  from  thirty-five 
to  fifty-five  minutes  developed  when  transferred  to  normal 
sea-water;  the  majority  disintegrated  in  the  course  of  the  next 
twenty-four  hours.  But  the  greater  part  of  the  eggs  which 
had  been  taken  from  the  hypertonic  sea-water  with  KCN  after 
fifty-five  minutes  were  then  placed  not  in  normal  sea-water, 
but  in  hypertonic  sea-water  (50  c.c.  sea-water +8  c.c  2§  m 
NaCl),  which  this  time  contained  no  potassium  cyanide. 
Samples  of  these  eggs  were  transferred  to  normal  sea-water 
20,  30,  40,  50,  and  60  minutes  later.  Of  these  eggs  taken  out 
of  the  hypertonic  sea-water  after  forty  minutes,  some  5  per  cent 
developed,  of  those  taken  out  after  fifty  minutes  some  30  per 
cent,  while  practically  all  of  those  transferred  after  sixty  min- 
utes developed,  though  some  already  showed  the  effects  of 
over-exposure  by  abnormal  cleavage.  This  experiment,  which 
was  repeated  several  times  with  the  same  result,  shows  that  the 


106    Artificial  Parthenogenesis  and  Fertilization 


hypertonic  solution  remains  practically  ineffective  in  the  pres- 
ence of  a  minute  quantity  of  potassium  cyanide,  but  that  the 
eggs  are  not  in  the  least  injured  by  the  latter  in  so  short  a  time. 
It  can  be  directly  shown  that  the  hypertonic  solution  is 
effective  only  in  the  presence  of  free  oxygen,  by  simply  expelling 
the  air  from  the  hypertonic  solution.     But  this  experiment 
can  very  easily  miscarry  on  account  of  the  interference  of  a 
troublesome  source  of  error.     Usually  the  hypertonic  solution 
can  be  freed  as  far  as  possible  from  oxygen  by  passing  through 
it  for  several  hours  a  current  of  scrupulously  purified  hydrogen. 
Then  a  drop  or  two  of  eggs  are  placed  in  the  solution.     And 
herein  lies  the  source  of  error.     On  opening  the  flask,  of  course 
some  oxygen  enters  it,  and  for  a  short  time  the  hypertonic 
solution  is  acting,  not  in  the  absence,  but  in  the  presence,  of 
some  oxygen.     Now  obviously  only  a  little  oxygen  is  sufficient 
to  maintain  the  processes  of  oxidation  which   underlie  the 
development  of  the  egg.     I  had  already  noticed  this  eighteen 
years  ago  in  my  first  experiments  upon  the  necessity  of  oxygen 
for  normal  cleavage.     But,  as  already  mentioned,  the  eggs 
need  to  remain  only  a  short  time — some  thirty  to  fifty  minutes 
— in  the  hypertonic  solution  after  membrane  formation,  and  it 
is  clear  that  in  so  short  a  time  the  introduction  of  a  little  oxygen 
into  the  hypertonic  solution  may  easily  frustrate  the  whole 
experiment.     I  reduced  this  risk  by  opening  the  stopper  of  the 
flask  with  the  aid  of  a  skilled  assistant  for  only  about  a  second 
and  for  a  distance  of  only  a  millimeter  in  order  to  introduce 
the  eggs.    Before,  during,  and  immediately  after  the  opening  a 
very  strong  current  of  hydrogen  was  passed  through  the  flask. 
Negative  experiments,  i.e.,  ones  in  which  the  hypertonic  solu- 
tion caused  a  few  or  many  of  the  eggs  to  develop,  even  after 
the  passing  of  a  current  of  hydrogen,  do  not  prove  much  ;^  but 

1  They  only  show  that  too  much  oxygen  was  present  inadvertently  in  the 
hypertonic  solution.  I  indicated  this  source  of  error  in  my  first  note  upon  the 
subject:  Loeb,  "On  the  Necessity  of  the  Presence  of  Free  Oxygen  in  the  Hyper- 
tonic Sea-Water  for  the  Production  of  Artificial  Parthenogenesis,"  University  of 
California  Publications,  Physiology,  III,  39,  1906. 


Action  of  the  Hypertonic  Solution  107 


on  the  other  hand,  those  experiments  are  important  in 
which  the  hypertonic  solution  remains  ineffective  in  the  ab- 
sence of  oxygen,  but  regains  its  powers  if  oxygen  be  afterward 

admitted. 

The  eggs  of  S.  purpuratus  were  treated  with  butyric  acid 
in  the  usual  manner,  and  all  formed  membranes.     These  eggs 
were  then  divided  between  two  flasks  containing  the  same 
hypertonic  solution.     Through  one  flask  bubbled  a  stream  of 
oxygen,  and  through  the  other  a  stream  of  hydrogen,  by  means 
of  which  it  had  been  previously  freed  from  oxygen.     The 
temperature  was  14°  C.     After  one  hour  the  eggs  were  trans- 
ferred to  normal  sea-water  (in  contact  with  air).     Practically 
all  the  eggs  which  had  been  in  the  oxygenated  hypertonic  sea- 
water  developed  into  larvae,  while  only  a  small  number  of  larvae, 
amounting  to  perhaps  one-half  of  1  per  cent  of  the  eggs,  devel- 
oped from  those  eggs  which  had  been  in  the  hypertonic  sea-water 
that  was  free  from,  or  more  strictly  poor  in,  oxygen.     The 
remaining  eggs  disintegrated  in  the  manner  characteristic  of 
eggs  that  have  undergone  artificial  membrane  formation  without 
exposure  to  hypertonic  sea-water.     I  now  wished  to  convince 
myself  that  the  eggs  which  go  to  pieces  after  exposure  to  hyper- 
tonic sea-water  that  is  free  from  or  poor  in  oxygen  do  develop, 
if  they  are  exposed  afterward  in  the  same  hypertonic  solution 
to  the  air.     To  this  end  not  all  of  the  eggs  were  removed  after 
the  conclusion  of  the  above  experiment  from  the  hypertonic 
solution  out  of  which  the  oxygen  had  been  driven,  but  some  of 
them  were  left  in  this  hypertonic  solution.     This  time,  however, 
the  latter  was  exposed  to  the  air.     At  intervals  of  14,  26,  36, 
46,  56,  and  116  minutes  samples  of  the  eggs  were  transferred  to 
normal  sea-water.     The  result  is  given  in  Table  X. 

From  this  experiment  we  can  with  certainty  draw  the 
conclusion  that  the  hypertonic  solution  in  these  experiments 
is  effective  only  when  it  contains  a  sufficient  quantity  of  free 
oxygen. 


108    Artificial  Parthenogenesis  and  Fertilization 

5.  It  appeared  interesting  to  determine  in  what  way  an 
increase  of  the  concentration  of  HO  ions  in  the  hypertonic  solu- 
tion would  increase  the  rapidity  of  its  action.  To  this  end 
not  sea-water,  but  a  neutral  mixture  of  m/2  NaCl,  KCl,  CaCl2, 
and  MgCl2  (in  the  proportion  in  which  these  salts  are  present 

TABLE  X 


Time  of  Exposure  of  the 

Eggs  to  Hypertonic 

Sea-Wateb 


Without 
Oxygen 


With 
Oxygen 


60  m 
60  m 
60  m 
60  m 
60  m 
60  m 
60  m 


iiutes 
nutes 
nutes 
nutes 
nutes 
nutes 
nutes 


+ 
+ 


0 
14 


+  26 
-i-  36 
+  46 
+  56 
+  116 


minutes 
minutes 
minutes 
minutes 
minutes 
minutes 
minutes 


Percentage  of  the  Eggs 

That  Developed  into 

Swimming  Larvae 


1 

2 

4 
30 
30 
90 

a  few 
no  larvae,  all  the 
eggs  died 


in  sea-water)  was  used.  To  each  50  c.c.  of  this  solution,  8  c.c.  of 
21  m  NaCl  solution,  also  neutral,  were  added  to  produce  the 
necessary  hy pert oni city;  then,  0,  0.1,  0.2,  0.4,  0.8,  and  1.6 
c.c.  of  N/50  NaOH  were  added  each  to  50  c.c.  of  this  mixture. 
The  eggs  of  a  >S^.  purpuratus  were  divided  among  these  solutions 
after  membrane  formation  had  been  produced  by  the  use  of 
butyric  acid.     Table  XI  gives  the  percentage  of  larvae  resulting. 


TABLE  XI 

Time  of  Exposure  to  the 
Hypertonic  Solution 

Amount  of  Alkali  Added  to  the  Neutral  Hypertonic 
Solution  in  c.c.  of  N/50  NaHO 

0 

0.1 

0.2 

0.4 

0.8 

1.6 

30  minutes 

0 

5% 
20 
25 
80 

a  few 
larvae 

10% 

80 

90 

90 

5% 

20 
50 
90 
90 

5% 

20 
80 
90 
90 

a  few 
larvae 

50% 

80 

90 

90 

a  few 

40  minutes 

larvae 
20% 
100 

50  minutes 

60  minutes 

90 

70  minutes 

90 

Action  of  the  Hypertonic  Solution  109 


The  larvae  were  crippled  by  too  long  an  exposure.  I  also 
found  that  one  could  even  obtain  larvae  if  a  trace  of  HCl  was 
added  to  the  hypertonic  solution.  I  have  not  yet  determined 
the  lower  limit  of  the  concentration  of  hydroxylions  in  the 
hypertonic  solution.^ 

6.  We  will  now  proceed  to  the  discussion  of  an  apparent 
contradiction  between  the  results  of  this  and  of  the  previous 
chapter.  In  the  latter  we  saw  that  the  eggs  can  be  made  to 
develop  after  membrane  formation  by  keeping  them  for  three 
hours  or  more  in  water  that  is  poor  in  oxygen,  or  by  preventing 
the  oxidations  by  the  addition  of  KCN.  In  this  chapter  we 
saw  that  the  eggs  after  membrane  formation  can  be  made  to 
develop  by  putting  them  afterward  into  hypertonic  sea-water 
for  some  thirty  to  fifty  minutes;  but  that  this^is  only  possible 
if  the  hypertonic  sea-water  contains  a  sufficient  amount  of  free 
oxygen.  How  are  these  apparently  contradictory  statements  to 
be  reconciled? 

The  essence  of  the  activation  of  the  unfertilized  egg  con- 
sists in  the  production  of  membrane  formation.  This  process 
is  certainly  not  one  of  oxidation,  since  it  can  take  place  in  the 
absence  of  oxygen  or  in  the  presence  of  KCN.  Perhaps  it 
depends  ultimately  upon  a  purely  physical  process  (such  as  tlie 
liquefaction  of  a  lipoid  or  the  dissociation  of  a  [hypothetical] 
lipoid-protein  combination). 

As  soon  as  this  process  has  taken  place,  development  sets 
in  in  the  egg.  Why  this  is  the  case,  we  do  not  at  present  know. 
But  at  the  same  time  this  process  leaves  the  egg  in  an  abnormal 
or  sickly  condition.  If  the  egg  begins  to  develop  while  in  this 
injured  condition,  it  disintegrates.  But  if  we  prevent  the 
egg  from  developing  for  some  hours,  by  depriving  it  of  ox>'gen, 
or  stopping  oxidations  by  the  addition  of  KCN,  the  egg  can 
develop  normally.     We  must  seek  the  reason  for  this  in  the 

1  Loeb,  "Zur  Analyse  der  osmotischen  Entwicklungserregung  unbefruchteter 
Seeigeleier,"  PflUger's  Archiv,  CXVIII,  197,  1907. 


110     Artificial  Parthenogenesis  and  Fertilization 

assumption  that,  while  the  oxidations  are  inhibited,  certain 
other  processes,  such  as  hydrolyses,  go  on  in  the  egg,  and  that 
these  processes  lead  to  the  formation  of  substances  which  now 
allow  the  egg  to  develop  without  disintegrating  in  this  process. 
But  the  same  result  can  be  obtained  more  quickly  if  we  modify 
the  process  of  oxidation  or  some  of  its  effects  by  putting  the 
eggs  for  a  short  time  into  a  hypertonic  solution. 

We  have  already'  mentioned  that  it  makes  no  difference  in 
principle  whether  the  eggs  are  first  caused  to  form  membranes, 
or  whether  they  are  first  placed  in  hypertonic  sea-water  and 
the  membrane  formation  is  instigated  afterward.^  The  only 
difference  between  the  two  procedures  consists  in  the  fact  that 
in  the  latter  case  the  eggs  must  remain  much  longer  in  the  hyper- 
tonic sea-water. 

7.  If  the  egg  is  treated  with  hypertonic  sea-water  before  the 
artificial  membrane  formation,  it  has  to  remain  about  twice 
as  long  in  the  hypertonic  solution  as  if  the  order  of  events  is 
reversed.  The  unfertilized  eggs  of  S.  purpuratus  were  put  into 
hypertonic  sea-water  (50  c.c.  sea- water +8  c.c.  2|  m  Ringer) 
and  portions  of  these  eggs  were  transferred  to  normal  sea-water 
after  30,  60,  90,  120,  150,  and  180  minutes.  Ten  minutes  after 
they  were  transferred  artificial  membrane  formation  was  called 
forth  by  butyric  acid.  The  eggs  which  had  been  30  and  60  min- 
utes in  the  hj'pertonic  solution  all  disintegrated  in  the  way 
characteristic  for  eggs  in  which  membrane  formation  has  been 
called  forth,  but  which  have  not  undergone  this  treatment 
with  hypertonic  sea-water,  A  few  of  the  eggs  that  had  been 
90  minutes  in  hypertonic  sea-water  developed.  Practically  all 
of  those  that  had  been  150  minutes  in  the  hypertonic  sea-water 
developed.  Those  eggs  which  had  only  undergone  the  treat- 
ment with'  the  hypertonic  solution  without  being  subsequently 
treated  with  butyric  acid  did  not  develop  into  larvae.     We 

1  For  practical  purposes  the  natural  order,  membrane  formation  followed  by 
hypertonic  solution,  is  preferable. 


Action  of  the  Hypertonic  Solution  ill 


have  already  stated  that  the  treatment  of  unfertilized  eggs  of 
S.  purpuratus  with  hypertonic  sea-water  alone  leads  to  the 
formation  of  larvae,  not  with  the  eggs  of  all  females,  but  with 
only  a  small  percentage. 

When  the  order  of  events  was  reversed  and  the  treatment 
with  hypertonic  sea-water  followed  the  artificial  membrane 
formation  all  the  eggs  developed  if  they  remained  in  the  sea- 
water  from  about  50  to  60  minutes.  The  reason  for  this  differ- 
ence is  easily  understood  if  we  compare  the  rate  of  oxidations 
before  and  after  membrane  formation.  After  membrane  forma- 
tion the  rate  of  oxidations  is  more  than  four  times  as  large  in  the 
egg  as  before  (chap.  xii).  With  this  fast  rate  of  oxidation  the 
eggs  need  remain  only  a  short  time  in  the  hypertonic  solution. 
If,  however,  we  put  the  eggs  in  the  hypertonic  solution  before 
the  membrane  is  formed,  they  have  to  stay  more  than  twice  as 
long  in  the  hypertonic  solution  because  the  rate  of  oxidations 
is  at  first  so  much  slower. 


XII 

THE  EFFECT  OF  THE  AGENCIES  OF  ARTIFICIAL  PAR- 
THENOGENESIS UPON  THE  OXIDATIONS.  THE  CY- 
TOLOGICAL  CHANGES  IN  THE  PARTHENOGENETIC 
EGG 


1.  The  experiments  thus  far  considered  have  shown  that  it 
is  possible  to  imitate  the  activating  effect  of  the  spermatozoon 
upon  the  egg  of  the  sea-urchin  approximately  by  submitting 
the  egg  to  two  different  processes.  The  first  process  consists 
in  calling  forth  the  membrane  formation  in  the  egg  by  a 
fatty  acid  (or  as  we  shall  see  later  by  a  number  of  other 
chemicals).  This  process  seems  to  be  the  essential  feature  in 
the  activation  of  the  egg,  since  it  suffices  to  set  in  motion  the 
whole  apparatus  of  nuclear  and  cell  division.  The  second 
process  has  only  a  corrective  effect,  since  the  membrane  forma- 
tion alone  leads  to  a  rapid  disintegration  of  the  egg,  unless  the 
temperature  is  very  low.  The  prevention  of  this  disintegration 
is  brought  about  by  the  second  process.  This  second  process 
consists  in  submitting  the  egg  for  a  short  period  to  a  hypertonic 
solution  containing  oxygen  (or  for  a  longer  period  to  sea-water 
free  from  oxygen). 

We  will  now  consider  the  effects  of  these  two  processes  upon 
the  oxidations  in  the  egg,  and  afterward  gain  an  insight  into 
the  cytological  changes  produced  in  the  egg  by  these  agencies. 

We  will  first  consider  the  effect  of  the  membrane  formation. 
The  reader  will  remember  that  I  had  shown  that  the  disinte- 
gration of  the  egg  which  follows  the  artificial  membrane  forma- 
tion at  room  temperature  (if  the  egg  is  not  treated  also  with  a 
hypertonic  solution  or  lack  of  oxygen)  can  be  retarded  for  a 
long  time  if  the  egg  is  kept  without  oxygen.     From  this  fact  I 

113 


114     Artificial  Parthenogenesis  and  Fertilization 


concluded  that  the  artificial  membrane  formation  increases  the 
rate  of  oxidations  in  the  egg. 

The  correctness  of  this  surmise  was  proven  by  0.  Warburg, 
who  compared  the  effect  of  the  artificial  membrane  formation 
upon  the  oxidations  in  the  egg  with  that  of  fertilization  by 
sperm.  The  ratio  was  10.5  for  eggs  fertilized  by  sperm  and 
9.0  for  eggs  after  artificial  membrane  formation.^  Hence  the 
effect  of  the  artificial  membrane  formation  upon  the  rate  of 
oxidations  was  nearly  the  same  as  that  of  fertilization  by  sperm. 
Warburg's  experiments  were  performed  on  the  egg  of  S.  lividus 
at  Naples.  Wasteneys  and  I  repeated  the  experiments^  on 
the  eggs  of  ^S.  purpuratus  at  Pacific  Grove. 

In  three  experiments  on  S.  purpuratus  the  ratio  of  oxygen 
consumption  between  unfertilized  and  fertilized  eggs  was 
1/6.87,  1/5.45,  and  1/5.60. 

A  comparison  of  the  ox>^gen  consumption  of  unfertilized 
eggs  before  and  after  membrane  formation  by  butyric  acid  gave 
in  one  experiment  a  ratio  of  1/4.72;  in  a  second  experiment  a 
ratio  of  1/4.28.  Since  this  figure  was  a  little  lower  than  the 
ratio  found  between  unfertilized  and  fertilized  eggs,  part  of  the 
eggs  of  the  same  females  were  utilized  to  determine  the  rate  of 
oxidation  in  the  unfertilized  and  fertilized  egg.  It  was  found 
to  be  1/4.55.  We  ma}'^  therefore  state  that  the  artificial  mem- 
brane formation  raises  the  rate  of  oxidations  to  approximately 
the  same  height  as  the  entrance  of  a  spermatozoon.  This 
confirms  the  conclusion  the  writer  had  drawn  concerning  the 
role  of  the  artificial  membrane  formation,  namely,  that  it  is  the 
essential  feature  in  the  activation  of  the  egg;  and  second,  that 
the  activation  consists  in  an  increase  in  the  rate  of  oxidations. 
The  question  then  arises.  In  which  way  can  the  artificial  mem- 
brane formation  increase  the  rate  of  oxidation?  We  may 
anticipate  here  what  will  be  proved  extensively  in  subsequent 
chapters,  that  the  membrane  formation  may  be  considered  as  a 

1  Warburg,  Zeitschr.  f.  physiol.   Chem.,  LXVI,  305,   1910. 
-  Loeb  and  Wasteneys,  Jour.  Biol.  Chem.,  XIV,  469,  1913. 


Artificial  Parthenogenesis  and  Oxidations       115 


superficial  cytolysis  or  a  cytolysis  of  the  cortical  layer  of  the 
egg.     It  is  obviously  this  cytolysis  and  no  subsequent  morpho- 
logical change  in  the  egg  which  causes  the  increase  in  the  rate 
of  oxidation.     We  can  cause  complete  cytolysis  of  the  eggs  of 
the  sea-urchin  by  adding  a  trace  of  saponin  to  the  sea-water. 
Wasteneys  and  I  measured  the  rate  of  oxidations  in  a  lot  of 
unfertilized  eggs  in  sea-water  and  found  that  they  consumed 
0. 15  mg.  O2  per  hour  at  15°  C.     Then  the  eggs  were  cytolyzed 
with  saponin  and  the  amount  of  oxygen  consumed  in  one  hour 
at  15°  C.  was  measured  again.     It  was  1 .07  mg.    The  cytolysis 
of  the  eggs  increased  the  rate  of  oxidation  700  per  cent,  as  much 
as,  or  possibly  a  trifle  more  than,  fertilization.     In  a  similar 
experiment  in  which  dilute  sea-water  was  used  for  cytolysis  and 
in  which  not  all  the  eggs  cytolyzed,  the  rate  of  oxidation  was 
increased  2 .  74  times  after  the  cytolysis.     The  cytolyzed  eggs 
are  no  longer  able  to  undergo  any  development  or  structural 
change.     It  is  therefore  obvious  that  the  increase  in  the  rate 
of  oxidation  after  membrane  formation  as  well  as  after  fertiliza- 
tion is  due  to  the  mere  cytolysis.     How  the  cytolysis  can  bring 
about  such  a  result  is  unknown. 

2.  The  greatest  interest  was  attached  to  the  question  as  to 
how  the  hypertonic  solution  affected  the  oxidations  in  the  eggs 
after  membrane  formation.  It  was  found  that  the  eggs  in 
which  the  artificial  membrane  formation  had  been  induced 
with  butyric  acid  consumed  oxygen  in  the  hypertonic  solution 
at  the  same  rate  as  in  normal  sea-water. 

Some  of  the  eggs  of  the  last-mentioned  exT3eriments  were 
also  used  to  compare  the  rate  of  oxidation  in  such  eggs  in  normal 
sea-water  and  in  hypertonic  sea-water  (50  c.c.  sea-water-f8  c.c. 

2imNaCl+KCl+CaCl2). 

Oxygen  consumption  of  unfertilized  eggs  after   artificial 

membrane  formation 

In  normal  sea-water 0-85  nig. 

In  hypertonic  sea-water 0 .  88  mg. 


116    Artificial  Parthenogenesis  and  Fertilization 


The  values  are  practically  identical.  The  eggs  treated 
with  the  hypertonic  solution  developed  after  being  put  in  normal 
sea-water. 

In  a  second  experiment  the  unfertilized  eggs  consumed  after 
membrane  formation  with  butyric  acid  in  normal  sea-water 
0 .  52  mg.  O2  in  65  minutes.  The  same  eggs  were  then  put  into 
hypertonic  sea-water  (50  c.c.  sea-water +8  c.c.  2 J  m  Ringer) 
and  consumed  here  in  65  minutes  0.54  mg.  O2.  The  tempera- 
ture was  in  both  cases  18°.  These  eggs  developed  after  being 
transferred  to  sea-water. 

In  another  experiment  the  unfertilized  eggs  consumed,  after 
the  artificial  membrane  formation  with  butyric  acid,  0 .  83  mg. 
in  60  minutes  in  normal  sea-water;  during  the  next  hour  they 
were  put  into  hypertonic  sea-water  and  consumed  in  60  minutes 
0 .  74  mg.  During  the  next  60  minutes  they  were  again  put  into 
normal  sea-water  where  they  consumed  0.70  mg.  O2  in  60 
minutes  (at  the  same  temperature).^ 

It  is  obvious  from  these  experiments  that  the  hypertonic 
solution  does  not  act  by  increasing  the  rate  of  oxidations.  This 
agrees  with  the  conclusion  we  reached  before,  that  the  mem- 
brane formation  is  the  real  activating  agent  while  the  hypertonic 
solution  acts  only  as  a  corrective. 

3.  Warburg^  states  that  if  the  eggs  of  S.  lividus  are  fertilized 
with  sperm  and  afterward  put  into  a  hypertonic  solution  the 
rate  of  oxidations  is  thereby  increased  300  per  cent.  Since 
Wasteneys  and  the  writer  found  that  the  hypertonic  solution 
of  the  concentration  required  for  artificial  parthenogenesis  does 
not  raise  the  oxidations  of  the  eggs  of  S.  purpuratus  after 
artificial  membrane  formation,  we  were  curious  to  know  whether 
such  a  hypertonic  solution  raises  the  rate  of  oxidations  in  the 
eggs  of  S.  purpuratus  fertilized  with  sperm.  Table  XII  gives 
the  result.^ 

1  Loeb  and  Wasteneys,  Jour.  Biol.  Chem.,  XIV,  469,  1913. 
-Warburg,  Zeitschr.  /.  physiol.  Chem.,  LX,  443,  1909. 
3  Loeb  and  Wasteneys,  oj}.  cit. 


Artificial  Parthenogenesis  and  Oxidations       117 


These  and  other  similar  experiments  show  that  the  rate  of 
oxidations  in  the  fertihzed  eggs  of  Strongylocentrotus  is  not 
altered  if  the  eggs  are  put  into  a  hypertonic  solution  of  that 
concentration  and  during  that  period  of  time  which  is  required 
in  the  method  of  artificial  parthenogenesis.  Only  if  the  ferti- 
lized eggs  remain  a  much  longer  period,  90  minutes  or  longer, 
in  the  hypertonic  sea-w^ater  is  the  rate  of  oxidation  altered — 
however,  not  increased  but  diminished. 

TABLE  XII 


Fertilized  Eggs  in 

Exp.  I.     Normal  sea-water 

Hypertonic  sea- water  (50  c.c.  sea- 
water+8  c.c.  2^   m    NaCl+KCl-{- 

CaCL) 

Exp.  II.     Normal  sea- water 

Hypertonic  sea-water  (50  c.c.  sea- 
water+8c.c.    2\    m    NaCl+KCIi- 

CaCla) 

Exp.  III.     Normal  sea-water 

Hypertonic  sea-water  (50  c.c.  sea- 
water +8  c.c.  2\  m  NaCl+KCl  + 
CaCh) 


Duration  of 
Experiment 


75  min. 


75 
90 


90 
60 


60 


Oxygen 
Consumption 


0.87  mg. 


0.86 
0.60 


0.52 
0.55 


0.59 


It  may  also  be  stated  that  Wasteneys  and  I  tried  the  effect 
of  hypertonic  solutions  of  various  concentrations  upon  the 
fertilized  eggs  of  S.  purpuratus.  The  result  was  always  the 
same:  The  hypertonic  solution  did  not  increase  the  rate  of 
oxidations  in  the  fertilized  egg  of  ^5^.  purpuratus,  no  matter 
how  high  the  concentration  was  raised,  as  Table  XIII  shows. 
Temperature  18°  C. 

It  is  obvious  that  the  increase  in  the  concentration,  even 
beyond  that  used  in  the  experiments  on  artificial  partheno- 
genesis, does  not  increase  the  rate  of  oxidations  in  the  fertilized 
eggs  of  S.  purpuratus. 

4.  This  result  creates  an  apparent  difficulty,  namely,  why  the 
hypertonic  solution  does  not  produce  its  corrective  efi'ect  upon 
the  egg  (after  artificial  membrane  formation)  in  the  absence  of 


118    Artificial  Parthenogenesis  and  Fertilization 


ox^^gen  or  if  the  oxidations  in  the  egg  are  retarded  through  the 
presence  of  KCN  (see  chap.  x).  This  difficulty  is,  however,  not 
real,  if  we  assume  that  the  corrective  effect  of  the  h^-pertonic 
solution  consists  in  the  production  of  a  substance  or  a  condition 
in  the  egg  which  cures  it  from  the  threatening  disintegration 
or  in  the  destruction  of  a  substance  or  a  condition  which  causes 
this  dishitegration.  Such  effects  might  be  produced  by  a 
slight  modification  of  the  character  of  the  processes  of  oxidation 
without  its  being  necessary  that  the  rate  of  oxidation  be  altered. 

TABLE  XIII 


Fertilized  Eggs  in 


Exp.  I.     Normal  sea- water 

50  c.c.  sea-water+4  c.c.  2^  m  (NaCl+ 

KCl+CaCU 

Exp.  II.     Normal  sea-water 

50  c.c.  sea-water +12  c.c.  2|  m  (NaCl  + 

KCl+CaCL) 

Exp.  III.     Normal  sea-water 

50  c.c.  sea-water +  16  c.c.  2f  m  (NaCl  + 
KCl+CaCL) 


Duration  of 
Experiment 


60  min. 

60 
60 

60 
60 

60 


Oxj'^gen 
Consumption 


1.30  mg. 


1.27 
1.33 

1.53 
1.33 

1.57 


Meyerhof  raised  the  objection  that  the  ratio  between  the  con- 
sumption of  oxygen  and  the  production  of  heat  was  the  same  in 
eggs  in  hypertonic  solutions  as  in  normal  sea-water.  But  I 
do  not  think  that  this  objection  speaks  against  my  hypothesis, 
since  it  is  only  necessary  that  the  hypertonic  solution  lead  to  the 
formation  of  a  by-product  in  a  very  minute  quantity,  while  this 
by-product  is  not  formed  when  the  eggs  develop  in  normal  sea- 
water.  Such  a  slight  qualitative  modification  of  the  oxidations 
could  very  well  exist  without  resulting  in  a  noticeable  alteration 
of  the  ratio  between  oxidations  and  heat  production  in  the  egg. 
It  is  also  possible  that  the  effects  of  the  products  of  oxidation 
are  different  in  hypertonic  and  normal  sea-water,  and  that  this 
determines  the  corrective  effect  of  the  hypertonic  solution. 

Such  an  assumption  would  also  enable  us  to  understand  why 
the  withdrawal  of  oxygen  for  a  longer  period  of  time,  namely. 


Artificial  Parthenogenesis  and  Oxidations       119 


about  three  hours,  or  the  addition  of  some  KCN  to  the  sea- 
water  for  the  same  period  of  time  after  the  artificial  membrane 
formation,  can  act  as  a  substitute  for  the  40  to  60  minutes  of 
treatment  of  the  eggs  with  the  hypertonic  solution.  It  is  con- 
ceivable that  the  hydrolytic  processes  which  continue  to  go  on  in 
the  egg  after  the  retardation  of  oxidations  lead  to  the  formation 
of  a  substance  (or  to  a  condition)  which  at  the  normal  rate  of 
oxidation  could  not  be  formed  (or  arise),  or  if  formed  would  be 
rapidly  destroyed,  and  which  acts  similarly  as  the  substance 
formed  in  a  much  shorter  time  through  the  oxidations  if  the 
amount  of  water  in  the  egg  is  diminished. 

5.  The  idea  that  the  curative  or  corrective  effect  of  the 
hypertonic  solution  consists  in  the  formation  in  the  egg  of  a 
substance  which  remedies  the  danger  of  disintegration  follow- 
ing artificial  membrane  formation  is  supported  by  another  fact. 
We  have  already  stated  that  the  treatment  of  the  egg  with  tlie 
hypertonic  solution  may  precede  the  artificial  membrane  forma- 
tion. But  the  writer  succeeded  in  showing  last  winter  that  if 
the  eggs  are  once  treated  with  the  hypertonic  solution  they  are 
permanently  immune  against  the  disintegration  which  follows 
artificial  membrane  formation.^ 

Unfertilized  eggs  of  S.  purpuratus  were  put  for  2  and  2^ 
hours  into  hypertonic  sea-water  (50  c.c.  sea-water -|-8  c.c.  2|  m 
Ringer).  Some  of  them  were  treated  about  ten  minutes  later 
with  butyric  acid,  and  the  majority  of  them  developed  int 
larvae.  Others  were  treated  with  butyric  acid  24  hours, 
48,  and  72  hours  later.  Those  treated  24  hours  later  with 
butjoic  acid  developed  also  and  about  as  well  as  those  treated 
immediately.  After  48  hours  a  great  many  eggs  were  dead, 
but  those  that  were  still  alive  or  had  not  suffered  too  much  still 
developed  into  larvae  when  treated  with  butyric  acid.  .Aiter 
three  days  ahnost  all  the  eggs  were  dead,  but  those  that  were 
still  intact  segmented  and  developed  into  swimming  larvae  after 
the  butyric-acid  treatment. 

1  Loeb,  Jour.  Exper.  Zool.,  XV,  201.  1913. 


O 


120    Artificial  Parthenogenesis  and  Fertilization 

Hence  by  a  treatment  with  a  hypertonic  solution  for  a 
sufficient  length  of  time  unfertiUzed  eggs  retain  as  long  as  they 
live  the  quality  of  being  immune  against  the  disintegration 
which  follows  artificial  membrane  formation.  What  is  the 
nature  of  this  alteration?  Warburg  found  that  in  unfertilized 
eggs  without  membranes  the  rate  of  oxidation  is  increased  by 
hypertonic  solutions,  and  we  were  able  to  confirm  his  observa- 
tion. This  suggested  the  idea  that  possibly  a  treatment  of  such 
eggs  with  a  hypertonic  solution  Avould  raise  their  rate  of  oxida- 
tion permanently  and  that  this  might  be  the  cause  of  their 
immunity  against  the  disintegrating  processes  following  arti- 
ficial membrane  formation.  This  was  a  priori  not  very  probable 
since  we  found  that  the  corrective  effect  of  the  hypertonic  solu- 
tion after  artificial  membrane  formation  in  the  egg  of  S.  pur- 
pur  atus  is  not  due  to  an  increase  in  the  rate  of  oxidations. 

6.  We  were  therefore  anxious  to  see  if  the  rate  of  oxidation 
caused  by  the  treatment  of  unfertilized  eggs  of  S.  purpuratus 
(without  membrane  formation)  was  permanently  maintained. 
For  this  purpose  the  consumption  of  oxygen  in  an  unfertilized 
lot  of  eggs  of  S.  purpuratus  was  measured  at  18°  C.  in  four  suc- 
cessive periods. 

Oxygen 
Consumption 
in  90  Minutes 

In  normal  sea-water 0-30  mgm. 

In  hypertonic   sea- water   (50  c.c.   sea-water +9  c.c.   2^  m 

NaCl+KCl+CaCU 0-67  mgm. 

In  normal  sea-water  half  an  hour  later 0.51  mgm. 

In  normal  sea-water  twenty-one  hours  later 0.48  mgm. 

While  it  is  obvious  that  these  eggs  continue  to  show  an 
increased  rate  of  oxidation  for  at  least  22  hours,  the  rate  is 
very  much  lower  than  after  fertilization  or  after  the  artificial 
membrane  formation.  Through  the  treatment  of  the  eggs  in 
the  above-mentioned  experiment  the  rate  was  increased  2.2 
times   and   the  next  day  it  had  fallen  to    1.6  times.    This 


Artificial  Parthenogenesis  and  Oxidations       121 

increase  is  so  small  that  it  is  not  likely  to  be  responsible  for  the 
corrective  action  of  the  hypertonic  solution. 

It  is  more  probable  that  the  treatment  of  the  egg  with  the 
hypertonic  solution  brings  about  a  permanent  change  in  the 
egg  by  which  it  can  now  undergo  the  process  of  membrane 
formation  at  any  time  without  danger  of  subsequent  disinte- 
gration. This  irreversibility  of  the  corrective  effect  of  the 
hypertonic  solution  would  be  intelligible  on  the  assumption 
that  it  is  due  to  the  formation  of  a  definite  substance  which  is 
retained  by  the  egg  and  w^hich  is  a  preventive  against  the 
disintegration  following  membrane  formation. 

7.  We  may  here  discuss  parenthetically  a  hypothesis  put 
forward  by  R.  Lillie^  concerning  the  nature  of  the  corrective 
action  of  the  hypertonic  solution.  Lillie  suggests  that  the 
essential  feature  in  the  act  of  fertilization  is  an  increase  in 
the  surface  permeability  of  the  egg  of  which  the  membrane 
formation  is  the  consequence;  and  that  the  effect  of  the  "after- 
treatment  with  a  hypertonic  solution  is  to  bring  the  permeability 
again  to  normal." 

We  are  able  to  investigate  the  relative  permeability  of 
fertilized  and  unfertilized  eggs  for  bases  with  the  aid  of  a 
color  test,  and  this  color  test  shows  that  both  unfertilized  as 
well  as  fertilized  eggs  are  permeable  for  weak  and  impermeable 
for  strong  bases.^  As  far  as  acids  are  concerned,  the  fact 
found  by  the  writer  that  weak  acids  like  CO^  and  the  fatty 
acids  cause  membrane  formation  in  the  unfertilized  egg  shows 
that  such  an  egg  must  be  permeable  for  these  substances. 
McClendon^  has  published  experiments  which  he  thinks  sup- 
port Lillie's  view  of  an  increased  ion-permeability  of  the  egg 
after   membrane    formation,    but    the    writer    does    not    feel 

1  R.  LUlie,  Jour.  Morphol.,  XXII,  695,  1911;  Am.  Jour.  Physiol.,  XXYII. 
289,  1911. 

2  0.  Warburg,  Zeitschr.  f.  physiol.  Chem.,  LXVI,  305,  1910;  N.  Harvey.  Jour. 
Exper.  Zool.,  X,  507,  1911. 

3  McClendon,  Am.  Jour.  Physiol.,  XXVII,  240,  1910. 


122    Artificial  Parthenogenesis  and  Fertilization 

that  McClendon's  interpretation  of  the  experiment  is  the  only 
one  admissible. 

McClendon  investigated  the  electrical  conductivity  of  mi- 
fertilized  and  fertilized  eggs  and  found  that  the  conductivity 
of  the  egg  is  increased  by  fertilization.  He  concludes  that 
this  proves  an  increased  permeability  of  the  egg  for  ions, 
but  the  same  result  would  be  produced  if  in  consequence  of 
fertilization  or  membrane  formation  the  degree  of  electroljiiic 
dissociation  in  the  surface  film  of  the  egg  should  be  increased. 
The  egg  or  its  surface  film  must  be  considered  as  a  non-aqueous 
phase  and  the  conductivity  in  this  phase  depends  among  others 
upon  the  degree  of  electrolytic  dissociation  of  the  electrolytes 
dissolved  in  it.  It  is  quite  conceivable  that  so  considerable  a 
change  in  the  cortical  layer  as  that  taking  place  in  membrane 
formation  might  influence  the  degree  of  solubility  or  of  electro- 
lytic dissociation  in  the  surface  film  of  the  egg. 

McClendon  made  also  the  interesting  observation  that  "if 
fertilized  and  imfertilized  eggs  of  Arhacia  are  placed  in  an  iso- 
tonic sugar  solution  containing  little  sea-water,  through  which 
a  current  of  gradually  increasing  density  is  passed,  the  unferti- 
lized eggs  begin  to  disintegrate,  at  their  anode  ends,  sooner  than 
the  fertilized  eggs."  He  interprets  this  as  "indicating  that  the 
fertilized  eggs  are  more  permeable  to  anions,  which  therefore 
accumulate  in  them  to  a  less  extent,  or  the  fertilized  eggs  are 
more  permeable  to  electrolytes,  which  therefore  have  passed 
out  into  the  sugar  solution  to  a  greater  extent,  and  therefore 
the  current  passes  through  them  less,  than  in  the  case  of  the 
unfertilized  eggs."  I  believe  that  the  phenomenon  described 
by  McClendon  is  not  sufficiently  understood  to  lend  itself  to 
conclusions  concerning  the  permeabihty  of  the  egg.  But  these 
questions  do  not  concern  us  here  so  much  as  the  action  of  the 
hypertonic  solution. 

The  idea  that  the  treatment  of  the  egg  with  the  hj^ertonic 
solution  serves  the  purpose  of  restoring  the  increased  permeability 


Artificial  Parthenogenesis  and  Oxidations       123 


of  the  egg  to  its  normal  limit  might  fit  those  cases  in  which 
the  hypertonic  treatment  follows  the  artificial  membrane  forma- 
tion, but  it  is  difficult  to  understand  how  it  can  be  made  to 
harmonize  ^vith  the  fact  that  the  treatment  of  the  egg  with  a 
hypertonic  solution  is  equally  efficient  if  it  precedes  the  artificial 
membrane  formation  by  twenty-four  hours  or  more.  More- 
over, we  shall  see  lat«r  on  that  bases  act  somewhat  like  acids 
in  inducing  membrane  formation,  and  that  the  eggs  must  also 
be  exposed  to  the  hypertonic  solution.  In  this  case  both 
agencies,  the  base  and  the  hypertonic  solution,  may  be  combined 
to  act  simultaneously  upon  the  egg  with  good  results.  This 
fact  seems  also  unintelligible  on  the  assumption  that  the  bases 
increase  the  permeability  of  the  egg  (to  allow  some  of  its  con- 
tents to  escape),  while  the  hj^ertonic  solution  has  the  opposite 
effect.  And  finally  one  does  not  understand  that  a  hypertonic 
solution  of  NaCl  (e.g.,  50  c.c.  m/2  NaCl-fl2  or  16  c.c.  2J  m 
NaCi),  which  acts  as  a  corrective  agent  upon  the  eggs  after 
membrane  formation,  should  diminish  their  permeability,  since 
all  our  experience  indicates  that  such  a  solution  injures  the  sur- 
face of  the  egg  and  increases  its  permeability. 

II 

8.  We  will  now  briefly  mention  some  cytological  points 
worthy  of  discussion.  E.  Hindle  has  investigated  the  cytologi- 
cal changes  in  the  eggs  of  S.  purpuratus  which  had  been  treated 
with  butyric  acid  and  subsequently  with  a  hypertonic  solution. 
He  found  that  the  changes  taking  place  in  such  eggs  were  almost 
identical  with  those  which  take  place  after  the  entrance  of  a 
spermatozoon.     Hindle^  gives  the  following  description: 

The  interval  (about  20  minutes)  between  the  transference  of  the 
eggs  from  butyric  acid  to  normal  sea- water  and  their  subsequent 
treatment  with  hypertonic  salt  solution  is  characterized  by  the  altera- 
tions in  the  appearance  of  the  cytoplasm  and  nucleolus,  and  the 

1  E.  Hindle,  Archiv /.  Entwicklungsmechanik,  XXXI,  145,  1911. 


124     Artificial  Parthenogenesis  and  Fertilization 

subsequent  development  of  a  perinuclear  zone,  as  described  above. 
The  nucleus  then  commences  to  grow  and  faint  radiations  can  some- 
times be  seen  extending  from  the  perinuclear  zone  into  the  surrounding 
cytoplasm. 

During  immersion  in  the  hypertonic  solution  there  are  no  apparent 
changes  beyond  a  shght  reduction  of  the  clear  zone  of  hyaloplasm 
surrounding  the  nucleus. 

After  the  eggs  are  put  back  into  normal  sea-water  the  internal 
changes  resulting  in  the  first  cleavage  follow  each  other  in  quick  suc- 
cession. The  first  change  noticed  is  an  increase  in  the  development  of 
the  perinuclear  zone,  followed  by  further  growth  of  the  nucleus.  ?^Iean- 
while,  the  meshwork  of  chromatin  becomes  coarser  and  more  aggre- 
gated together  and  the  nucleolus  gradually  disappears.  This  stage 
is  succeeded  by  a  reduction  of  the  perinuclear  zone  together  with  its- 
radiations. 

About  half  an  hour  after  transference  to  normal  sea-water,  from 
one  pole  of  the  nucleus  a  definite  aster  begins  to  develop,  its  rays 
focusing  in  a  more  or  less  indistinct  centrosome  situated  on  the  nuclear 
membrane.^  By  division  of  the  centrosome  a  typical  amphiaster  is 
formed  in  the  nuclear  area  and  as  it  develops  the  nuclear  membrane 
disappears.  At  the  same  time  the  chromatin  assumes  the  form  of 
a  spireme,  which  subsequently  breaks  up  into  about  IS  long  and 
slender  chromosomes.  At  this  stage  it  is  impossible  to  clearly  dis- 
tinguish their  number,  but,  as  the  chromosomes  are  gradually  drawn 
into  the  equator  of  the  cleavage  amphiaster,  they  shorten  consider- 
ably and  become  quite  distinct  by  the  time  that  the  equatorial  plate 
is  formed. 

At  this  stage  we  have  made  numerous  counts  of  the  chromosomes 
and  invariably  found  it  in  the  neighborhood  of  18,  which  is  half  the 
number  that  is  normally  present  in  this  species. 

This  behavior  is  very  similar  to  the  one  found  in  the  egg 
after  fertilization  by  sperm. 

1  It  may  be  well  to  call  special  attention  to  the  fact  that  the  centrosomes  and 
astrospberes  are  not  formed  while  the  eggs  are  in  the  hypertonic  solution  but 
some  time  after  they  are  put  back  into  the  normal  sea-water.  Only  if  the  eggs 
remain  too  long  in  the  hypertonic  sea-water  centrosomes  and  cytasters  may  form 
while  the  eggs  are  in  the  hypertonic  sea-water,  not,  however  because  they  are  in 
this  solution  but  in  spite  of  it.  The  hypertonic  solution  allows  the  internal  process 
leading  to  the  formation  of  astrospheres  to  go  on  for  some  time.  This  led  to  the 
erroneous  idea  that  the  hypertonic  solution  was  the  direct  cause  of  the  formatioa 
of  centrosomes  and  astrospheres. 


Artificial  Parthenogenesis  and  Oxidations       125 


In  natural!}^  fertilized  eggs  a  distinct  aster  (cleavage  aster)  appears 
at  one  pole  of  the  nucleus,  its  rays  centering  in  a  clear  area  which  repre- 
sents a  diffuse  centrosome.  This  area  divides  and  the  two  halves 
move  apart  until  they  come  to  lie  at  opposite  sides  of  the  nucleus  and 
form  the  poles  of  a  typical  amphiaster  which  is  developed  in  the 
nuclear  region.  Meanwhile  the  chromatin  assumes  the  form  of  a 
spireme,  which  breaks  up  into  36  chromosomes  that  arrange  themselves 
about  the  equator  of  this  amphiaster  to  form  a  nuclear  spindle.  In 
the  chemically  fertilized  eggs  a  nuclear  spindle  arises  in  a  similar  way 
and  the  chromatin  assumes  the  form  of  a  spireme  preparatory  to 
breaking  up  into  chromosomes,  but,  instead  of  36,  only  18  of  these 
latter  bodies  appear.  The  subsequent  changes  are  identical  in  both 
kinds  of  eggs.  The  chromosomes  split  longitudinally  and  each  half 
moves  along  the  spindle  fibres  toward  its  respective  pole.  As  they 
approach  the  poles  the  chromosomes  swell  up  and  eventually  fuse 
together  to  form  a  single  nucleus  in  the  region  occupied  by  each  of  the 
diffuse  centrosomes.  Meanwhile  a  cell  wall  develops  between  the  two 
nuclei  dividing  the  cytoplasm  into  two,  and  finally  the  spindle  fibres 
disappear.  The  succeeding  processes  of  development,  both  internal 
and  external,  are  similar  in  both  naturally  and  chemically  fertilized 
eggs,  with  the  exception  that  at  each  succeeding  division  only  18  chro- 
mosomes appear  in  the  latter  instead  of  the  normal  number,  36.^ 

We  had  mentioned  that  the  eggs  which  formed  a  membrane 
upon  butyric-acid  treatment  begin  to  divide  and  may  go  through 
a  series  of  divisions  if  the  temperature  is  sufficiently  low. 
According  to  Hindle,  at  room  temperature  only  a  monaster  is 
formed  with  the  nucleus  as  a  center,  while  at  a  low  temperature 
a  typical  amphiaster  is  formed.  The  subsequent  changes  in  the 
latter  case  are  the  same  as  with  the  treatment  with  hypertonic 
sea-water. 

1  Hindle,  op.  cit. 


XIII 

THE  RELATIVE  PHYSIOLOGICAL  EFFICIENCY  OF 
VARIOUS  ISOSMOTIC  SOLUTIONS^ 

As  already  mentioned,  I  had  in  1900  published  the  obser- 
vation that  the  development  of  the  egg  of  Arhacia  can  be 
initiated  by  a  pure  sugar  solution.^  These  experiments  had 
already  shown  that  the  pure  sugar  solution  exerted  a  stronger 
osmotic  effect  than  it  should  theoretically. 

The  following  experiments  were  performed  upon  the  eggs  of 
S.  purpuratus  in  which  membrane  formation  had  been  previ- 
ously produced  by  treating  them  with  butyric  acid.  The 
eggs  were  placed  in  the  hypertonic  solution  some  ten  minutes 
after  membrane  formation,  and  hence  the  time  of  exposure 
is  longer  than  it  would  be  had  they  been  transferred  to  the 
hypertonic  solution  half  an  hour  after  membrane  formation. 
We  will  start  with  experiments  with  pure  sodium  chloride 
solution. 

The  experiments  with  pure  hypertonic  solutions  of  NaCl 
gave  results  that  are,  at  first  glance,  paradoxical;  for  the  eggs 
after  membrane  formation  can  tolerate  a  higher  concentration 
of  NaCl  solution  than  of  hypertonic  sea-water.  We  shall  see, 
however,  that  this  paradox  finds  a  simple  explanation.  The 
unfertilized  eggs  of  a  female  were  made  to  form  membranes  by 
treating  them  with  butyric  acid,  and  then  (about  ten  minutes 
later)  they  were  divided  among  50  c.c.  |  m  NaCl +3,  4,  5,  6, 
7,  8,  10,  12,  14,  and  16  c.c.  2^  m  NaCl;  some  of  the  eggs  were 
transferred  to  normal  sea-water  after  55,  90,  and  120 
minutes.     The  temperature  was  13°  C. 

1  Lioeb,  "Ueber  den  Unterschied  zwischen  isosmotischen  und  isotonischen 
Losungen  bei  der  kunstliclien  Parthenogenese,"  Biochem.  Zeitschr.,  XI,  144,  1908. 

2Loeb,  Am.  Jour.  Physiol,  IV,  178,  1900. 

127 


128    Artificial  Parthenogenesis  and  Fertilization 


If  we  compare  these  results  with  those  obtained  in  chapter 
xi,  Ave  find  that  50  c.c.  of  m/2  NaCl +5  c.c.  of  2|  m  NaCl 
produce  the  same  effect  as  50  c.c.  of  sea-water +4  c.c.  of 
2 J  m  NaCl;  in  other  words,  that  a  mixture  of  50  c.c.  of  J  m 
NaCl+1  c.c.  of  2J  m  NaCl  is  about  isotonic  with  sea-water. 

TABLE  XIV 


Composition  of  the  Solution 

Percentage  of  Eggs  That 

Developed  into  Larvae 

after  an  exposure  of 

55  min. 

90  min. 

120  min. 

50  c.c.  h  m  NaCl+  3  c.c.  2§  m  NaCl 

50  c.c.  ^  m  NaCl+  4  c.c.  2|  m  NaCl 

50  c.c.  ^  m  NaCH-  5  c.c.  2|  m  NaCl 

50  c.c.  ^  m  NaCl-h  6  c.c.  2^  m  NaCl 

50  c.c.  ^  m  NaCl+  7  c.c.  2h  m  NaCl 

50  c.c.  ^  m  NaCl+  8  c.c.  2|  m  NaCl 

50  c.c.  h  m  NaCl  +  10  c.c.  2^  m  NaCl 

50  c.c.  1  m  NaCl  +  12  c.c.  2^  m  NaCl 

50  c.c.  1  m  NaCl  +  14  c.c.  2i  m  NaCl 

50  c.c.  h  m  NaCl+16  c.c.  2^  m  NaCl 

0 

0 

0 

0 

2 

30 

80 

80 

80 

80 

0 
0 

0 

1 

50 

80 

90 

•  • 

•  • 

0 
0 

1 

5 

70 

90 

•  • 

•  • 

•  • 

•  • 

Now  whereas  a  mixture  of  50  c.c.  of  sea-water+10  c.c.  of 
2J  m  NaCl  injures  the  eggs  so  badly  in  60  minutes  that  only  a 
small  number  develop,  and  a  mixture  of  50  c.c.  of  sea-water + 
12  c.c.  of  2i  m  NaCl  kills  practically  all  eggs  with  artificial  mem- 
branes in  60  minutes,  in  a  mixture  of  50  c.c.  of  ^  m  NaCl +16  c.c. 
of  2i  m  NaCl  80  per  cent  of  the  eggs  develop  after  an  exposure 
of  55  minutes.  The  explanation  of  this  paradox  lies  in  the  fact 
that  the  NaCl  solution  was  practically  neutral,  while  the  sea- 
water  is  slightly  alkaline.  We  shall  see  in  a  later  chapter  that 
the  harmful  effect  of  hypertonic  solutions  upon  the  egg  is  much 
greater  if  they  are  slightly  alkaline  than  when  they  are  neutral; 
slightly  acid  hypertonic  solutions  are  still  less  injurious  than 
neutral  ones,  or,  in  other  words,  the  harmful  effect  of  hypertonic 
solutions  increases,  within  the  limits  considered,  with  the  con- 
centration of  the  hj^droxylions.  The  injurious  effect  of  these 
hypertonic  solutions  is  diminished,  if  not  absent,  in  a  solution 


Physiological  Efficiency  of  Isosmotic  Solutions     129 


which  is  free  from  oxygen:   hence  it  is  possible  that  there  is  a 
connection  between  these  two  facts. 

We  now  come  to  the  experiments  with  cane  sugar.  Accord- 
ing to  freezing-point  determinations  performed  by  Dr.  W.  E. 
Garrey,  the  sea-water  in  Pacific  Grove  lowers  the  freezing-point 
to  the  same  extent  as  a  0.54  m  NaCl  solution.  This  agrees 
pretty  well  with  the  above-mentioned  data.  Since,  according 
to  Kohlrausch  and  Holborn,  some  74  per  cent  of  the  molecules 
of  NaCl  are  dissociated  at  this  concentration,  a  0.94  m  cane- 
sugar  solution  is  theoretically  isosmotic  with  sea-water.  But  if 
experiments  are  performed  on  the  sea-urchin  egg  with  pure 
cane-sugar  solutions,  it  will  be  found  that  such  a  solution  acts 
as  though  it  were  not  isotonic  but  strongly  hypertonic.  0,  1,  2, 
3,  4,  5,  6,  7,  and  8  c.c.  of  2i  m  cane-sugar  solution  were  added 
each  to  50  c.c.  of  3/4  m  sugar  solution  and  the  unfertilized  eggs 
of  a  sea-urchin  divided  among  these  after  artificial  membrane 
formation.^  Temperature  12°  C.  The  eggs  remained  58  minutes 
in  the  solution.     The  percentage  of  larvae  formed  is  given  in 

Table  XV. 

TABLE  XV 


Composition  of  Solution 


50  c.c.  f  m  cane  sugar+0  c.c.  2|  m  cane  sugar, 
50  c.c.  f  m  cane  sugar4-2  c.c.  2^  m  cane  sugar. 
50  c.c.  f  m  cane  sugar +3  c.c.  2|  m  cane  sugar. 
50  c.c.  f  ra  cane  sugar+4  c.c.  2^  m  cane  sugar. 
50  c.c.  f  m  cane  sugar+5  c.c.  2h  m  cane  sugar. 
50  c.c.  J  m  cane  sugar +6  c.c.  2^  m  cane  sugar. 
50  c.c.  f  m  cane  sugar +7  c.c.  2h  m  cane  sugar. 
50  c.c.  f  m  cane  sugar+8  c.c.  2h  m  cane  sugar. 


Percentage  of 

Eggs  Developing 

into  Larvae 


0 
0 

a  few  larvae 
2 
20 
60 
98 
98 


Hence  a  mixture  of  50  c.c.  3/4  m+6  c.c.  2J  m  cane-sugar 
solution  acts  just  as  efifectively  as  a  mixture  of  50  c.c.  of  sea- 
water+8  c.c.  2i  m  NaCl  or  50  c.c.  i  m  NaCl+8  or  10  c.c.  2J  m 
NaCl.     A  mixture  of  50  c.c.  of  3  4  m+G  c.c.  2i  m  cane  sugar 

1  The  eggs  were  put  into  the  sugar  solution  a  few  minutes  after  membrane 
formation. 


130    Artificial  Parthenogenesis  and  Fertilization 


is,  however,  0.94  m,  or  isosmotic  with  sea-water.  But  this 
solution  acts  just  as  hj^pertonically  upon  the  sea-urchin  egg  as  a 
0.80  m  NaCl  solution,  i.e.,  a  solution  whose  osmotic  pressure 
is  about  50  per  cent  higher  than  that  of  a  0.94  m  cane-sugar 
solution. 

It  can  be  directly  shown  that  a  solution  of  cane  sugar, 
which  is  theoretically  isosmotic  with  sea-water,  is  actually 
hypertonic  for  the  sea-urchin  egg.  For  the  eggs  shrivel  in  such 
a  solution;  they  even  shrink  in  a  7/8  m  cane-sugar  solution;  in  a 
6/8  m  solution  they  retain  their  volume  and  in  a  5/8  m  solu- 
tion they  increase  in  size.  Eight  years  ago  I  also  observed  that 
medusae  (Polyorchis)  shrink  considerably  in  a  pure  cane-sugar 
solution  that  is  theoretically  isosmotic  with  sea-water. 

It  can  be  indirectly  shown  that  a  3/4  m  cane-sugar  solution 
is  about  the  concentration  that  is  isotonic  for  the  sea-urchin 
egg,  by  placing  sea-urchin  eggs  that  have  been  fertilized  with 
sperm  in  pure  cane-sugar  solutions  of  different  concentrations 
immediately  after  fertilization.  Experiments  of  this  description 
showed  that  in  a  6/8  m  cane-sugar  solution  the  first  cell  division 
occurs  in  all  the  eggs  of  purpuratus,  and  indeed  at  almost  the  same 
time  as  in  normal  sea- water;  while  in  5/8  and  7/8  m  solutions  its 
onset  is  delayed  and  occurs  in  only  a  few  eggs.  In  cane-sugar 
solutions  below  5/8  m  and  above  7/8  m  as  a  rule  not  a  single 
egg  divided.  In  6/8  m  cane-sugar  solution,  again,  the  cleavage 
did  not  go  beyond  the  four-  or  eight-cell  stage,  which  is  in 
accordance  with  the  experience  in  muscle.  But  if  the  eggs  are 
replaced  in  sea -water  they  develop  normally.  This  behavior 
of  the  egg  in  a  cane-sugar  solution  corresponds  to  the  behavior 
of  a  medusa  in  the  same  solution :  for  in  the  latter,  also,  as  well 
as  in  the  heart,  the  spontaneous  contractions  cease  in  a  pure 
cane-sugar  solution.^  These  observations  have  a  bearing  upon 
a  controversy  between  Delage  and  the  writer.  Delage  used  in 
his  experiments  on  artificial  parthenogenesis  cane-sugar  solu- 

1  Loeb,  Am.  Jour.  Physiol,  III,  384,  1900. 


Physiological  Efficiency  of  Isosmotic  Solutions     131 


tions  which  were  1.135  N,  e.g.,  a  mixture  of  40  c.c.  of  such 
a  cane-sugar  solution +10  c.c.  of  sea-water.  He  insisted  that 
these  solutions  were  isotonic  for  the  sea-urchin  eggs,  since 
their  molecular  concentration  was  equal  to  that  calculated 
from  the  depression  of  the  freezing-point  of  the  sea-water  which 
he  used.  Our  experiments  show  that  a  saccharose  solution 
which  is  theoretically  isosmotic  with  the  sea-water  is  neverthe- 
less physiologically  hypertonic.  Theoretically  a  0.94  m  sac- 
charose solution  is  isosmotic  with  the  sea-water  in  Pacific 
Grove.  Yet  a  0.94  m  saccharose  solution  was  not  physiologi- 
cally isotonic  with  sea-water,  but  acted  physiologically  like  a 
solution  equal  in  osmotic  pressure  to  a  mixture  of  50  c.c.  sea- 
water+8  c.c.  2i  m  NaCl,  i.e.,  a  hypertonic  solution.  This  dis- 
crepancy can  be  explained  partly  or  wholly  from  the  fact  that 
the  osmotic  pressure  of  cane-sugar  solutions  is  greater  than  we 
should  expect  from  their  molecular  concentration  (according 
to  the  direct  measurements  of  Lord  Berkeley  and  of  Morse). 
This  discrepancy  between  the  theoretical  and  real  osmotic  pres- 
sure of  cane-sugar  solutions  may  possibly  increase  with  the 

concentration. 

In  order  to  elucidate  whether  besides  the  merely  physical 
a  physiological  factor  was  also  involved  in  this  discrepancy 
between  theoretically  isosmotic  and  physiologically  isotonic 
solutions  some  experiments  ^vith  other  substances  were  tried. 

TABLE  XVI 


Constitution  of  the  Solution 
i_ 


50  c.c.  h  m  LiCl     +6  c.c.  2h  m  LiCl . 
50  c.c.  h  m  LiCl     +7  c.c.  2|  m  LiCl . 
50  c.c.  ^  m  KCl     +7C.C.  2|mKC. 
50  c.c.  h  m  KCl     +8  c.c.  2|  m  KCl 
50  c.c.  ^  m  MgCL+6  c.c.  2h  m  MgC 
50  c.c.  h  m  MgCl2+7  c.c.  2^  m  MgCh 
50  c.c.  h  m  CaCla  +6  c.c.  2h  m  CaCh 
50  c.c.  h  m  CaCU  +7  c.c.  2^  m  CaCL 


2 


2  ■ 


Percentage  of 

Eggs  That 

Produced  Larvae 


70 
1 

60 
50 
80 
20 
90 


132     Artificial  Parthenogenesis  and  Fertilization 


It  can  be  seen  from  this  that  solutions  of  various  substances 
which  are  all  theoretically  isosmotic  do  not  on  that  account 
possess  the  same  degree  of  physiological  activity.  It  is  there- 
fore necessary  to  differentiate  between  theoretically  isosmotic 
and  phj'siologically  isotonic  solutions.  The  two  values  are 
more  nearly  identical  for  the  red  blood  corpuscles  than  for  the 
sea-urchin  egg.  This  will  be  made  clear  by  the  following  table 
in  which  is  set  forth  the  optimum  concentration  of  solutions  of 
various  substances  with  regard  to  artificial  parthenogenesis.  For 
this  optimum  concentration  can  be  very  sharply  determined  b}' 
choosing  a  definite  length  of  exposure,  such  as  55  minutes  at 
about  15°  C. 

TABLE  XVII 

Optimum  Concentration   of  Solutions  of  Various  Substances  for 

Artificial  Parthenogenesis 


Constitution  of  the 
Solution 

Optimum  Con- 
centration in 
Grammolecules 

Dissociation  of 
the  Solution 

Osmotic  Pressure 

of  the  Solution  in 

Atmospheres 

Cane  sugar 

0.96  m 

1.04 

0.50 

0.49 

0.74 

0.79 

0.78 

•    • 

64 
70 

66 
71 

77 

21  53 

Grape  sugar 

23  33 

CaCl. 

25  57 

MgCU 

26  47 

LiCl 

27.59 
30  28 

NaCl 

KCl 

30.95 

Of  course  these  values  which  w^re  obtained  with  S.  purpur- 
atus  at  Pacific  Grove  do  not  apply  as  they  stand  to  any  given 
form  of  sea-urchin  in  any  given  locality.  We  have  no  explana- 
tion to  offer  for  these  discrepancies  except  in  the  case  of  sugars 
where  the  discrepancy  seems  to  find  its  explanation  in  the 
abnormal  physical  behavior  of  concentrated  solutions.  It  is 
possible  that  these  salts  modify  the  permeability  of  the  egg  in 
a  different  degree  or  sense  and  that  this  accounts  for  the  dis- 
crepancies between  calculated  and  observed  results. 


XIV 

CHEMICAL  CONSTITUTION  AND  RELATIVE  PHYSIOLOGI- 
CAL EFFICIENCY  OF  ACIDS^ 

In  my  first  experiments  upon  membrane  formation  I  found 
that  the  chemical  constitution  of  acids  is  of  great  importance 
with  regard  to  their  effect  upon  membrane  formation.     For 
whereas  carbonic  acid  and  the  weak  monobasic  fatty  acids  were 
very  effective,  the  strong  acids,  such  as  HCl,  HNO3,  and  H2SO4 
or  oxalic  acid,  had  so  Httle  effect  as  to  be  practically  useless  for 
these  experiments.     The  oxy-acids  were  effective,  but  not  to  tlie 
same  degree  as  the  monobasic  fatty  acids.     The  further  investi- 
gation of  the  relation  that  exists  between  constitution  and  effect 
seemed  to  be  full  of  promise,  as  it  was  to  be  expected  that  it 
would  give  some  information  upon  the  role  of  acids  in  membrane 
formation,  and  that  the  results  might  be  of  general  importance. 
The  following  was  the  procedure  adopted  in  the  experiments. 
The  eggs  were  first  freed  from  all  sea-water  by  being  twice 
washed  in  an  m/2  NaCl  solution.     They  were  then  put  into 
solutions  of  the  various  fatty  acids  in  m/2  NaCl  solution,  since 
it  was  necessary  to  make  the  acid  solution  isosmotic  with  the 
sea-water.     At  definite  intervals  a  portion  of  the  eggs  was  trans- 
ferred by  a  pipette  to  normal  sea-water,  and  the  percentage  of 
eggs  which  formed  membranes  determined. 

I  had  discovered  in  my  earliest  experiments  that  the  higher 
fatty  acids  had  more  effect  than  the  lower.  Hence  I  suspected 
that  the  activity  of  the  monobasic  fatty  acids  increased  with 
the  number  of  carbon  atoms.  The  results  of  one  of  a  series  of 
experiments  performed  to  decide  this  question  are  given  in 
Table  XVIII.     The  temperature  was  about  IS''  C. 

iLoeb,  B.ochem.  Zeitschr.,  XV,  254,  1909;  "An  Improved  .\[ethod  of  Arti- 
ficial Parthenogenesis,"  University  of  California  Publications,  Pliysiology.  II.  UOo, 
Untersuchungen,  p.  329,  Leipzig,  1906. 

133 


134    Artificial  Parthenogenesis  and  Fertilization 


The  first  vertical  column  of  Table  XVIII  gives  the  length  of 
time  that  the  eggs  had  remained  in  the  acid,  and  the  other 
vertical  columns  give  the  percentage  of  the  eggs  which  formed 
membranes  after  this  exposure  to  the  different  acids. 


TABLE  XVIII 


Length  of  Exposure  to 
N/1,000 

Formic 
Acid 

Acetic 
Acid 

Pro- 
pionic 
Acid 

Butyric 
Acid 

Caprylic 
Acid 

Nonylic 
Acid 

1  minute 

0 
0 
0 
0 
0 

n% 

30 

90 

100 

0 
0 
0 

i% 
5 
60 

•  •   ■   • 

•  •   •   • 

0 
0 

iV% 

20 

50 

75" 

•   •   •   • 

0 

h% 
10 
40 
90 
95 
100 

10% 
80 
100 

100% 

1|  minutes 

2  minutes 

2  J  minutes 

3  minutes 

3^  minutes 

4  minutes 

4|  minutes 

5  minutes 

It  will  be  seen  that  the  greater  the  number  of  carbon  atoms 
in  the  acid  molecule  the  shorter  is  the  time  which  is  necessarv 
to  cause  membrane  formation  in  a  definite  percentage  of  eggs. 
This  result  is  intelligible  on  the  assumption  that  those  acids 
which  diffuse  most  rapidly  into  the  egg  call  forth  membrane 
formation  in  the  shortest  time.  This  behavior  of  the  acids  is 
analogous  to  that  of  the  alcohols  whose  narcotic  and  haemolytic 
activity  increases  also  for  the  same  series  with  increase  of  the 
number  of  carbon  atoms.^  In  the  alcohols,  however,  the  in- 
crease of  activity  is  much  quicker  than  that  found  by  us  for 
the  acids,  for  each  member  of  the  series  is  about  two  or  three 
times  as  effective  as  the  preceding  one. 

Although  the  question  of  the  influence  of  the  concentration 
of  the  acid  upon  its  effect  is  not  so  intimately  connected  with 
our  subject,  one  example  may  be  noted  here  for  the  sake  of 

completeness.     I   quote   two   sets   of   experiments,   one   with 

I 

1  Fuliner  and  Neubauer,  "Hamolyse  durch  Substanzen  homologer  Reihen," 
Arch.  f.  exper.  Path.  u.  Pharm.,  LVI,  333,  1907;  Overton,  Studien  ueber  Narkose, 
Jena,  1901. 


Physiological  Efficiency  of  Acids 


135 


butyric  acid,  the  other  with  benzoic.     As  usual,  the  acid  was 
diluted  with  half  grammolecular  sodium  chloride  solution. 


TABLE  XIX 


Length  of 
Exposure  to — 


^  minute . 

1  minute .  . 
1^  minutes 

2  minutes . 
2-2"  minutes 

3  minutes . 

4  minutes . 

5  minutes. 

6  minutes . 

7  minutes . 


Butyric  Acid 


3/5,000 

N 


0 
0 
0 

2% 

3 

5 
30 
40 
80 
90 


5/5.000 

N 


0 

0 
48% 
60 
95 


6/5,000 

N 


0 

14% 
9.5 
100 


7/5,000 

N 


5% 
100 


8/5,000 

N 


5% 
100 


10/5,000 

N 


5% 
100 


TABLE  XX 


Length  of  Exposure 

Benzoic  Acid 

to — 

12/50,000  N 

18/50,000  N 

24/50,000  N 

h  minute 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 

Wo 
60 

100 

1  minute 

30% 

1^  minutes 

100 

2  minutes 

3  minutes 

4  minutes      

.5  minutes 

6  minutes 

7  minutes 

8  minutes 

As  a  rule  membrane  formation  cannot  be  elicited  by  the 
use  of  butyric  acid  at  a  lower  concentration  than  3/5,000  N. 
Even  at  concentrations  of  3/5,000  N  butyric  acid  is  usually 
without  effect.  When,  however,  the  effective  value  is  attained, 
the  optimum  concentration  is  also  quickly  reached.  The  same 
is  true  of  benzoic  acid,  only  in  this  case  the  effective  value  is 
much  lower  than  for  Ijutyric  acid,  being  18/50,000  N. 


136     Artificial  Parthenogenesis  and  Fertilization 


Before  going  farther,  we  should  point  out  that  the  minimal 
value  of  concentration  for  an  acid  to  prove  effective  is  not  quite 
the  same  for  eggs  of  different  females.  In  my  experiments  upon 
the  causation  of  development  of  sea-urchin  eggs  by  means  of 
the  blood  serum  of  warm-blooded  animals,  it  has  been  an  ever- 
recurring  observation  that  these  experiments  only  succeed  for 
a  part  of  the  eggs  of  a  female,  unless  the  eggs  are  previously 
sensitized  by  a  treatment  with  SrCU  (see  chap,  xviii).  I 
regard  the  variation  in  permeability  of  the  eggs  of  different 
females  to  acids  or  blood  as  responsible  for  these  individual  vari- 
ations. The  mass  of  the  eggs  also  exerts  some  influence.  If 
too  many  eggs  are  placed  in  the  solution,  the  stated  mass  of 
acid  is  not  enough. 

Overton  observes  that  the  narcotic  effect  of  the  dihydric 
alcohols  is  much  weaker  than  that  of  the  monohydric  alcohols. 
I  find  that  the  monobasic  acids  of  the  former  series  exhibit  a 
much  weaker  effect  with  regard  to  membrane  formation  than  the 
corresponding  members  of  the  series  of  acids  of  the  monatomic 
alcohols.  I  may  quote  as  evidence  experiments  with  ox^^pro- 
pionic  and  oxybutyric  acids. 


TABLE  XXI 


Length  of  Exposure 

OxYPROPioNic  Acid 

^-Oxybutyric  Acid 

N/500 

4/500  N 

N/500 

2/500  N 

4/500  N 

1  minute 

0 
0 
0 
0 
0 

0 

2% 
60 
90 

0 
0 

8% 

5% 
100 

100% 

2  minutes 

3  minutes 

4  minutes 

^  minutes 

•    •    •    •    • 

*    •    '    ■    • 

It  will  be  seen  from  a  comparison  between  this  and  the  pre- 
ceding table  that  /3-oxybutyric  acid  possesses  only  a  quarter  of 
the  efficiency  of  butyric  acid,  and  that  propionic  acid  is  more 
than  four  times  as  effective  as  lactic  acid.     The  oxy-acids  also 


Physiological  Efficiency  of  Acids 


137 


show  increasing  effectiveness  with  the  number  of  carbon  atoms; 
for  jS-oxybutyric  acid  is  about  twice  as  active  as  oxypropionic 
acid.  A  comparison  of  oxybenzoic  with  benzoic  acid  showed 
that  the  latter  is  ten  times  as  effective  as  the  former. 

Perhaps,  however,  the  influence  of  the  constitution  of  the 
acid  is  most  clearly  demonstrated  by  a  comparison  of  the  effect 
of  i3-oxybutyric  acid  and  oxyisobutyric  acid,  ^-oxybutyric 
acid  has  more  than  four  times  the  effect  of  oxyisobutyric  acid. 
This  is  probably  connected  with  the  fact  that  while  /3-ox>^butyric 
acid  has  the  same  carbon  chain  as  butyric  acid,  oxyisobutyric 
acid  has  a  branched  carbon  chain. 

Experiments  upon  di-  or  polybasic  acids  prove  a  trial  of 
patience,  as  one  cannot  with  certainty  rely  upon  success. 
Whereas  propionic,  butyric,  and  valerianic  acids  cause  mem- 
brane formation  in  all  eggs  of  *S.  purpuratus  practically  without 
exception  (provided  that  the  concentration  of  acid  and  length 
of  exposure  are  correctly  chosen),  the  di-  and  polybasic  organic 
acids  do  not  affect  the  eggs  of  all  females.  Hence  these  acids 
behave  Uke  foreign  blood  sera,  as  regards  membrane  formation. 
This  analogy  indicates  that  the  dibasic  acids  have  a  weaker 
effect  than  the  monobasic  acids,  because  they  enter  the  egg 
more  slowly.  For  obviously  the  degree  of  permeability  of  the 
egg  differs  in  various  females.  In  the  next  table  I  have  put 
together  results  obtained  from  the  eggs  of  an  especially  '' favor- 
able" female  with  oxalic,  succinic,  tartaric,  and  citric  acids. 


TABLE  XXII 


Length  of  Exposure  to— 


1  minute . 

2  minutes 

3  minutes 

4  minutes 

5  minutes 

6  minutes 


1.7/500  N 
Oxalic  Acid 


/o 


0 

15^- 
20 
90 
90 


7  500  X 

Succinic 

Acid 


0 
0 
0 
0 

2% 


7  500  N 

Tartaric 

Acid 


lO^c 
100 


4  500  N 
Citric  Acid 


0 

0 
10% 
60 
80 
90 


138    Artificial  Parthenogenesis  and  Fertilization 

In  order  to  make  the  above  discrepancies  comprehensible, 
we  must  remember  how  many  variations  of  constitution  come 
into  play  in  this  experiment.  We  saw  first  that  the  effect  of 
the  acid  increases  with  the  number  of  carbon  atoms,  secondly 
that  the  entry  of  an  HO-group  has  the  opposite  effect,  and 
thirdly  that  the  "linear"  coupling  of  the  carbon  atoms  is  more 
effective  than  the  "branched"  (ox^^butyric  and  oxyisobutyric 
acids).  All  these  and  other  conditions  of  constitution  prob- 
ably come  under  consideration  in  the  explanation  of  this  appar- 
ently irregular  acid  effect  of  the  last  table.  The  number  of 
acids  investigated  is  not  sufficient  for  a  detailed  analysis. 

It  is  even  truer  of  the  mineral  than  of  the  dibasic  organic 
acids  that  they  are  able  to  cause  membrane  formation  only  in 
the  eggs  of  some,  but  not  all  females.  With  sulphuric  acid  in 
particular  I  have  never  yet  been  able  to  evoke  membrane 
formation  in  the  sea-urchin  egg;^  I  have  done  so  occasionally 
with  HNO3  and  HCl,  but  not  always.  The  most  favorable 
results  that  I  have  obtained  with  these  two  acids  are  summed 
up  in  Table  XXIII. 

TABLE  XXIII 


Length  of  Exposure  to — 


1  minute. 

2  minutes 

3  minutes 

4  minutes 

5  minutes 

6  minutes 

7  minutes 

8  minutes 

9  minutes 
10  minutes 


N/500  HCl  N/500  HNO, 


0 

0 

^% 

10 
20 
30 
80 

80 
80 
90 


0 

10% 
80 
90 
100 


2/500  N 
HNO3 


5% 
90 
100 
100 


3/500  N 
HNO3 


90% 


Usually,  however,  the  results  are  more  of  the  kind  given 
for  comparison  in  Table  XXIV. 

1  It  seems  to  be  a  general  experience  that  sulphates  diffuse  less  easily  into 
living  cells  than  chlorides. 


Physiological  Efficiency  of  Acids 


139 


In  this  case  then  the  paradoxical  result  is  obtained  that 
one-thousandth  normal  butyric  acid  is  more  effective  in  causing 
membrane  formation  than  one-twelfth  normal  hydrochloric 
acid!  A  blind  opponent  of  the  dissociation  theory  could  wish 
for  no  more  striking  material  than  that  here  adduced.  Yet 
it  would  be  a  serious  mistake  to  make  use  of  these  results 
against  the  theory  of  electrolytic  dissociation. 

TABLE  XXIV 


Length  of  Exposure 
to— 

15/500  N  HCl 

40/500  N  HCl 

2  minutes 

3  minutes 

4  minutes 

5  minutes 

6  minutes 

0 

\7o 
1 

5 

30 

20% 
20 

20 

The  disagreement  of  the  facts  here  mentioned  with  the  dis- 
sociation theory  is  only  an  apparent  one  and  finds  its  solution 
in  the  consideration  of  the  following  two  facts.  In  the  first 
place,  with  regard  to  the  causing  of  membrane  formation,  only 
the  mass  of  acid  that  has  actually  entered  the  egg  comes  into 
consideration;  and  secondly,  the  velocity  with  which  the  different 
acids  enter  the  egg  is  a  function  of  their  chemical  constitution. 

If  this  is  true,  we  should  find  indications  that  the  connections 
mentioned  above  between  the  constitution  and  physiological 
effect  of  acids  in  reality  show  relations  between  constitution 
and  the  rapidity  of  absorption  of  the  acids  by  the  egg.  We 
will  produce  two  proofs  of  this,  one  indirect  and  the  other  direct. 
The  indirect  evidence  consists  in  the  fact  that  the  effects  of  the 
homologous  alcohols  in  the  experiments  of  Overton,  Fiihner 
and  Neubauer  are  analogous  to  the  effects  of  the  fatty  acids 
in  ours.  Now  Hans  Meyer  as  well  as  Overton  has  pointed  out 
that  the  activity  of  the  alcohols  runs  parallel  to  their  coefficient 
of  partition  between  lipoid  and  water.  The  relative  physio- 
logical activity  of  the  alcohols  must  therefore  be  determined 


140     Artificial  Parthenogenesis  amd  Fertilization 

chiefly  by  the  relative  velocity  of  their  absorption  by  the  cell, 
and  analogy  urges  a  similar  supposition  in  the  case  of  the  acids. 

The  direct  proof  is  as  follows.  If  it  is  true  that  the  weak 
monobasic  fatty  acids  are  more  effective  for  the  purpose  of 
membrane  formation  than  the  strong  mineral  acids,  for  the 
very  reason  that  the  former  are  more  quickly  absorbed  by  the 
egg,  it  would  be  expected  that  the  fatty  acids  are  more  injurious 
to  the  egg  and  kill  it  more  quickly  than  the  mineral  acids; 
for  in  order  to  kill  the  egg  the  acids  must  enter  it.  Now  this 
can  be  very  easily  tested  by  placing  the  eggs  of  the  same  female 
in  different  acids  (diluted  with  |  m  NaCl  solution),  and  deter- 
mining how  long  they  must  remain  in  the  various  acids  to  lose 
their  faculty  of  being  fertilized  and  of  developing. 

Unfertilized  sea-urchin  eggs  were  therefore  put  into  a  N/12 
solution  of  HCl  (in  |  m  NaCl  solution),  and  transferred  from 
this  to  normal  sea-water  every  half-minute,  when  a  sample  of 
them  was  fertilized  with  sperm.  Only  a  few  had  formed 
membranes  as  a  result  of  treatment  with  HCl,  and,  as  usual, 
these  all  went  to  pieces.  However,  those  eggs  which  had  been 
up  to  three  minutes  in  N/12  HCl  and  had  formed  no  membranes 
formed  them  upon  the  addition  of  sperm  and  developed  to 
swimming  larvae.  The  addition  of  sperm  caused  development 
in  20  per  cent  of  the  eggs  which  had  been  four  minutes  in  the 
HCl  solution  (and  which  had  formed  no  membranes  after  trans- 
ference to  normal  sea-water),  and  even  after  five  minutes' 
exposure  to  the  N/12  HCl  solution,  10  per  cent  of  the  eggs  could 
still  be  fertilized  and  caused  to  develop  by  sperm.  It  is  scarcely 
necessary  to  mention  that  weaker  concentrations  of  HCl  were 
much  less  injurious. 

Before  we  turn  to  the  experiments  upon  the  toxicity  of  the 
monobasic  fatty  acids,  I  must  once  again  remind  the  reader 
that  the  eggs  form  no  membrane  so  long  as  they  are  in  the  lower 
fatty  acids,  but  only  after  they  have  been  transferred  to  the 
(weakl}'  alkaline)  sea-water;  and  moreover  that  the  membrane 


Physiological  Efficiency  of  Acids 


141 


formation  does  not  occur  after  transference  to  normal  sea-water 
if  the  eggs  have  been  too  long  in  the  fatty  acid  solution.  Why 
this  should  be  so  I  cannot  tell;  possibly  too  much  fatty  acid 
enters  the  egg  and  the  latter  can  no  longer  form  a  membrane. 
The  following  table  gives  a  clear  picture  of  this  fact.  The  acid 
used  was  butyric. 

TABLE  XXV 

Percentage  of  Eggs  Which  Formed   Membranes  after  Exposure 

TO  Butyric  Acid 


Length  of  Exposure  to — 


1  minute . 

2  minutes 

3  minutes 

4  minutes 

5  minutes 


N/500 


100% 

100 

100 


2/500  N 


100% 

100 

1 


1 

2 

0 


3/500  N 


/o 


100^^ 

10 

0 

0 

0 


4  500  N 
Butyric  Acid 
in  m,  2  XaCl 


100% 

10 

0 

0 

0 


It  can  be  seen  from  this  table  that  unfertilized  sea-urchin 
eggs  can  no  longer  form  membranes,  if  they  remain  longer 
than  two  minutes  in  a  3/500  N  solution  of  butyric  acid  fin  h  m 
NaCl  solution).  If  sperm  now  be  added  to  such  eggs  after  they 
are  transferred  to  normal  sea-water,  no  egg  is  fertilized  and  none 
develops.  I  thought  at  first  that  it  depended  upon  a  reversible 
acid  effect  and  that  the  eggs  would  be  able  to  recover  after  a 
long  stay  in  sea-water.  But  that  is  not  the  case.  As  a  control, 
the  eggs  of  the  same  female  were  then  placed  in  a  N/50  solution 
of  HCl,  in  which  they  remained  four  minutes.  Not  one  formed 
a  membrane  after  transference  to  normal  sea-water.  But 
upon  the  addition  of  sperm  40  per  cent  of  these  eggs  were  ferti- 
lized and  developed  in  a  perfectly  normal  fashion.  We  have 
seen  that  benzoic  acid  is  much  better  for  causing  membrane 
formation  than  butyric  acid.  We  should  expect  that  it  is 
correspondingly  more  injurious.  This  is  also  the  case.  Eggs 
were  placed  in  a  N/500  benzoic  acid  solution.  Every  minute 
some  of  the  eggs  were  transferred  to  normal  sea-water.     The 


142     Artificial  Parthenogenesis  and  Fertilization 


eggs  transferred  from  the  benzoic  acid  after  one  minute  all 
formed  a  fertilization  membrane.  But  those  eggs  which  had 
remained  three  minutes  or  more  in  the  solution  of  benzoic 
acid  formed  no  membranes.  But  the  eggs  could  no  longer  be 
fertilized  with  sperm. 

The  objection  may  be  raised  against  these  experiments  that 
the  eggs  are  not  killed  by  the  fatty  acid,  but  only  made  imper- 
meable to  spermatozoa.  In  order  to  test  this  objection,  eggs 
were  first  fertilized  with  sperm  and  then  exposed  to  the  action 
of  the  acid  mentioned.  Fertilized  eggs  which  had  been  longer 
than  two  minutes  in  a  4/500  N  butyric-acid  solution  were  unable 
to  develop  after  transference  to  sea-water.  We  feel  justified 
therefore  in  regarding  it  as  certain  that  the  influence  of  the 
chemical  constitution  of  the  acid  upon  its  physiological  action 
is  to  be  referred  to  the  velocity  of  its  diffusion  into  the  egg. 
(The  latter  influence  is  perhaps  asserted  in  the  sense  that  the 
velocity  of  absorption  of  acid  into  the  egg  cell  increases  with  the 
increase  of  the  coefl&cient  of  partition  of  the  acid  for  oil  and 
water.) 

We  must,  however,  give  up  the  idea  that  the  physio- 
logical action  of  the  acids  is  determined  by  the  diffusion  of 
the  hydrogen  ion  into  the  egg.  Were  that  so,  the  activity 
of  the  acid  ought  to  correspond  to  the  concentration  of  free 
hydrogen  ions,  which  is  certainly  not  the  case,  as  the  inefl&ciency 
of  the  strong  acids  shows.  Hence  these  experiments  also 
furnish  proof  that  the  acids  enter  the  egg  cells  in  the  form  of 
undissociated  molecules.  In  my  earlier  publications^  (1905)  I 
had  already  been  led  to  the  conclusion  that  in  the  causation  of 
membrane  formation  by  acids  it  is  not  the  hydrogen  ion  but 
the  undissociated  molecules  which  come  into  play.  That  the 
anions  of  the  acids  do  not  diffuse  as  such  into  the  egg  is  shown 
by  the  fact  that  the  addition  of  the  salt  of  a  fatty  acid,  such  as 
sodium  acetate,  or  sodium  butyrate,  to  the  sea-water  causes  as 

1  Loeb,   University  of  California  Publications,  Physiology,  II,  May,  1905. 


Physiological  Efficiency  of  Acids  143 

a  rule  no  membrane  formation.  This  means  that  the  hitter 
must  depend  upon  the  diffusion  of  the  undissociated  acid  mole- 
cule. These  facts  support  the  idea  that  only  undissociated 
molecules  and  not  ions  diffuse  into  the  cell.  This  should 
be  taken  into  consideration  by  those  who  maintain  that  the 
effect  of  fertilization  consists  in  an  increased  permea})ility  of 
the  egg  for  ions. 

A  fact  which  agrees  well  with  the  above  statement  is  that 
carbonic  acid  is  especially  effective  for  membrane  forma- 
tion; I  discovered  this  in  my  earlier  experiments/  and  it  has 
been  confirmed  by  Godlewski.^  Carbonic  acid  is  a  very  weak 
acid. 

Now  it  is  found  that  the  eggs  form  no  membranes  upon 
treatment  with  the  lower  fatty  acids  while  they  are  in  the  acid, 
but  only  after  they  are  transferred  to  normal  sea-water.  This 
holds  for  formic,  acetic,  propionic,  butyric,  valerianic,  and 
capronic  acids.  Heptylic,  caprylic,  nonylic,  and  caprinic  acids, 
however,  behave  otherwise,  for  the  eggs  form  membranes  while 
they  are  in  the  solution  of  these  higher  acids.  The  explanation 
of  this,  1  believe,  is  to  be  found  in  the  fact  that  the  higher  mono- 
basic fatty  acids  are  but  little  soluble  in  water  and  very  soluble 
in  the  cell.  Hence  the  higher  fatty  acids  are  rapidly  absorbed 
by  the  cell,  and  the  sea-water  is  practically  free  from  acid. 
Hence  it  is  no  longer  necessary  to  transfer  the  eggs  into  sea- 
water  free  from  acid.  It  can  be  shown  that  membrane  forma- 
tion is  prevented  by  hydrogen  ions  because  no  membrane  for- 
mation occurs  if  the  eggs  are  placed,  after  exposure  to  butyric 
acid,  in  sea-water  to  which  some  mineral  acid  has  been  added. 
For  if  sea-urchin  eggs  are  transferred  after  treatment  with 
butyric  acid  to  50  c.c.  of  sea-water-f  1 .5  or  2  c.c.  of  N/10  HCl, 
as  a  rule  membrane  formation  no  longer  takes  place.  We 
have  mentioned  above  that  the  eggs  form  membranes  while  in 

1  Loeb,  Untcrsuchungen,  p.  34iS;  University  of  California  Publications,  Physiol- 
ogy, II.  1905. 

2  E.  Godlewski,  Archiv  f.  Entwicklungsmechanik,  XXVI,  27S,  1908. 


144     Artificial  Parthenogenesis  and-  Fertilization 

sea-water  that   contains   benzol;    but  if  HCl  is  added  to  this 
sea-water,  this  membrane  formation  does  not  take  place/ 

If  the  eggs  are  transferred  from  the  butyric  acid  solution, 
not  into  sea-water,  but  into  a  neutral  mixture  of  NaCl,  KCl,  and 
CaCL,  often  no  proper  membrane  is  formed.  But  if  the  eggs 
are  put  into  an  alkaline  mixture  of  NaCl,  KCl,  and  CaCU, 
proper  membranes  are  formed  by  the  egg. 

The  experiments,  however,  lead  to  still  another  unexpected 
result.  If  the  eggs  are  transferred  from  the  acid  solution  into 
the  neutral  mixture  of  NaCl,  KCl,  and  CaCU,  they  neither 
develop  nor  disintegrate.  They  appear  rather  to  return  into 
the  resting  condition  in  which  they  can  be  fertilized  with  sperm. 
I  have  fertilized  such  eggs  with  sperm  even  after  two  days, 
and  could  evoke  development. 

The  following  experiment  also  shows  that  such  eggs  remain 
in  the  resting  condition  if  they  are  transferred  from  the  acid 
.solution  to  the  neutral  mixture  of  NaCl,  KCl,  and  CaCU. 
If  the  eggs  are  placed,  after  treatment  with  the  fatty  acid,  in 
sea-water  or  in  an  alkaline  solution  of  NaCl,  KCl,  and  CaCl2 
(in  which  they  form  a  perfect  membrane),  a  further  short  treat- 
ment with  a  neutral  hypertonic  solution  is  sufficient  to  cause 
all  the  eggs  to  develop  into  larvae.  In  such  an  experiment  it 
was  only  necessary  to  leave  the  eggs  from  20  to  50  minutes  in 
a  neutral  hypertonic  solution  (50  c.c.  NaCl+2.2  c.c.  KC1  + 
1.5  c.c.  CaCla,  all  m/2,  +9  c.c.  2J  m  NaCl),  to  cause  all  the 
eggs  to  develop  into  larvae.  The  eggs,  however,  which  had 
been  transferred  from  the  acid  solution  into  the  neutral  solu- 
tion of  NaCl,  KCl,  and  CaCL,  could  generally  not  be  made  to 
develop  even  by  120  minutes'  exposure  to  the  neutral  hyper- 
tonic solution.  This  again  shows  that  these  eggs  were  really 
in  the  resting  condition.  These  facts  are  a  new  proof  for  the 
statement  made  in  a  previous  chapter,  that  the  acid  causes  the 
development  of  the  egg  only  indirectly  through  the  membrane 
formation. 

1  Loeb,  op.  cit. 


Physiological  Efficiency  of  Acids  145 


Why  is  it  that,  thougli  the  strong  mineral  acids  do  occasion- 
ally work,  as  a  rule  they  fail  ?  It  has  already  been  mentioned 
that  if  they  produce  any  effect,  membrane  formation  does 
not  set  in  while  the  eggs  are  in  the  acid  solution,  but  only  after 
they  have  been  transferred  to  normal  sea-water.  It  is  possi})l(> 
that  the  mineral  acids  only  cause  membrane  formation  indi- 
rectly, their  immediate  effect  being  to  liberate  from  its  salts 
some  fatty  acid  contained  in  the  surface  of  the  egg,  and  that 
then  the  fatty  acid  thus  liberated  instigates  membrane  forma- 
tion. This  hypothesis  is  supported  by  the  following  obser- 
vation. If  2  or  3  c.c.  of  N/10  HCl  are  added  to  50  c.c.  of 
sea-water,  and  unfertilized  eggs  placed  in  this  solution,  no 
membranes  are  as  a  rule  formed  by  the  eggs  after  transference 
to  normal  sea-water.  A  similar  result  is  obtained  if,  instead  of 
HCl,  2  or  3  c.c.  of  N/2  sodium  butyrate  are  added  to  the  sea- 
water.  But  if  both  HCl  and  sodium  butyrate  are  added  simul- 
taneously to  the  sea-water,  the  eggs  do  form  a  membrane 
when  transferred  from  this  to  normal  sea-water.  In  this  case 
butyric  acid  is  produced  and  diffuses  into  the  egg.  And  there 
is  no  reason  for  supposing  that  a  similar  reaction  may  not  take 
place  when  HCl  comes  into  contact  with  the  surface  of  the  egg; 
for  it  might  liberate  here  one  of  the  higher  fatty  acids  contained 
in  combination  in  the  surface  of  the  egg. 

It  may  also  be  mentioned  that  the  addition  of  a  slightly 
effective  mineral  acid,  such  as  HCl,  to  butyric  acid  does  not 
influence  the  activity  of  the  latter.  I  have  performed  many 
experiments  of  the  kind,  to  see  how  far  HCl  can  replace  the 
fatty  acid.  It  appears  that  HCl  can  only  enter  as  substitute 
for  a  small  amount  of  the  fatty  acid;  perhaps  some  acid  is 
combined  with  the  proteins  of  the  egg  (especially  of  the  chorion), 
and  only  this  amount  of  fatty  acid  can  be  replaced  by  HCl. 
If  more  is  added,  everything  proceeds  as  though  Xhv  fatty  acid 
concerned  were  in  solution  alone.  The  only  effect  of  the  sur- 
plus of  H  ions  is  that  the  confluence  of  the  drops  in  membrane 


146     Artificial  Parthenogenesis  and  Fertilization 


formation  does  not  proceed  so  readily.  All  this  harmonizes 
with  the  view  that  it  is  not  the  hydrogen  ions  but  only  the  undis- 
sociated  acid  which  diffuses  into  the  egg  and  that  the  fatty  acids 
diffuse  much  more  quickly  than  the  mineral  acids  which  may 
not  enter  at  all. 

We  may  inquire  whether  the  causation  of  membrane  forma- 
tion is  a  chemical  or  purely  a  physical  effect  of  the  acid.  I 
asked  Dr.  Hagedoorn  to  determine  the  temperature  coefficient 
of  membrane  formation  by  means  of  acids.  He  found  that 
it  is  about  2  for  a  difference  of  temperature  of  10°  C,  which 
therefore  indicates  a  chemical  reaction.  The  procedure  was 
to  measure  the  minimum  time  which  eggs  must  remain  in  a 
mixture  of  50  c.c.  of  acid +2. 5  c.c.  of  N/10  butjTic  acid  to  cause 
membrane  formation  in  95  per  cent  of  the  eggs.  It  turned  out 
that  this  time  is  twice  as  long  at  10°  C.  as  at  20°  C.  This 
result  was  confirmed  by  other  determinations  at  different  tem- 
peratures. 

All  the  experiments  mentioned  in  this  chapter  were  made  on 
the  eggs  of  the  Californian  sea-urchin  S.  purpuratus. 


XV 

THE  ACTIVATION  OF  THE  UNFERTILIZED  EGG  BY  BASES 

1.  The  writer  foundjn  1907  that  strong  bases  are  able  to 
cause  the  unfertiHzed  egg  to  de\'elop,  but  that  in  some 
respects  their  effect  differs  from  that  of  acids.  While  only  a 
short  treatment  with  acid  suffices  to  induce  membrane  forma- 
tion and  while  this  action  is  not  prevented  by  KCN,  the  strong 
bases  must  act  on  the  egg  a  comparatively  long  time  before 
they  can  cause  it  to  develop.  And,  moreover,  their  effect  is 
prevented  by  lack  of  oxygen  or  by  the  presence  of  KCN.^ 

Like  the  acids,  the  bases  cause  development  through  a 
modification  of  the  surface  of  the  egg,  but  in  the  case  of  bases 
the  resulting  membrane  is  as  a  rule  only  a  fine  gelatinous  layer, 
such  as  is  formed  through  the  influence  of  fatty  acids  in  the  egg 
of  Arhacia,  although  occasionally  a  typical  fertilization  mem- 
brane is  formed.     The  bases  act  therefore  in  one  respect  like 
the  acids,  inasmuch  as  both  call  forth  a  typical  or  atji^ical 
membrane  formation  and  this  membrane  formation  is  the  essen- 
tial part  of  artificial  parthenogenesis.     In  order  to  cause  the 
unfertilized  eggs  of  Arhacia  to  develop  by  bases,  the  following 
procedure  was  found   to  be   effective.     The  eggs  of   Arhacia 
are  put  into  50  c.c.  m/2  (NaCl+KCl+CaCy+O.S  c.c.  N/10 
NH4OH  for  twenty-five  minutes,  at  a  temperature  of  about  22°  C. 
From  here  they  are  transferred  directly  to  a  neutral  h3'pertonic 
solution,  50  c.c.  m/2    Ringer +8  c.c.  2i  m  Ringer,  for   fifteen 
minutes,  and  then  they  are  put  back  into  normal  sea-water. 
In  this  case  a  large  number  of  eggs  develop  into  larvae,  many 
of  which  are  perfectly  normal.     It  will   be  found  that  onl\' 
those  eggs  develop  into  larvae  which  form  the  gelatinous  film— 

1  Loeb,  "Ueber  die  allgemeineu  Methoden  der  kuiisUichen  Parthenogenese  " 
PflU&er's  Archiv,  CXVIII,  572,   1907. 

147 


148     Artificial  Parthenogenesis  and  Fertilization 


the  membrane.  This  membrane  does  not  usually  form  while 
the  eggs  are  in  the  alkaline  solution  but  afterward  cither  in  the 
hypertonic  solution  or  sometimes  even  later. ^ 

It  is  very  important  that  the  eggs  should  not  remain  too 
long  in  the  h\'pertonic  solution.  The  time  they  must  remain  in 
the  hypertonic  solution  varies  according  to  the  length  of  time 
the  eggs  remained  in  the  NH4OH  solution.  Eggs  that  were 
kept  in  the  above-mentioned  alkaline  solution  for  ten  minutes 
developed  best  when  they  were  exposed  to  the  hypertonic 
solution  for  twenty-four  minutes;  while  eggs  that  were  in  the 
alkaline  solution  for  thirty  minutes  developed  best  when  put 
for  fifteen  minutes  into  the  hypertonic  solution.  This  observa- 
tion finds  its  possible  explanation  in  the  fact  that  the  hyper- 
tonic solution  also  causes  an  increase  in  the  rate  of  oxidation 
of  the  unfertilized  egg,  and  in  this  respect  resembles  the  action 
of  the  alkahne  solution.  It  is  therefore  not  surprising  that  the 
two  solutions  can  act  as  a  partial  substitute  for  each  other.  If 
the  eggs  remain  only  a  few  minutes  too  long  in  the  hypertonic 
solution,  they  suffer  considerably;  if  they  are  taken  out  of  the 
hypertonic  solution  too  soon,  they  will  not  develop. 

2.  A  comparison  of  the  relative  efficiency  of  various  alkalies 
for  the  causation  of  artificial  parthenogenesis  furnished  the 
result  that  weak  bases  like  NH4OH  are  much  more  effective 
than  strong  bases  like  NaOH,  KOH,  or  tetraethylammonium- 
hydroxide.  To  three  solutions  of  50  c.c.  m/2  Ringer  were 
added  0.3  c.c.  N/'IO  NH4OH,  0.3  c.c.  N/iONaOH,  and  0.3  c.c. 
N/10  tetraethylammoniumhydroxide  respectively.  Unferti- 
lized eggs  of  Arbacia  were  put  into  these  solutions  for  twenty- 
six  minutes  and  were  then  transferred  to  the  hypertonic  Ringer 
solution  (50  c.c.  m/2  Ringer +8  c.c.  2|  m  Ringer).  They 
remained  here  for  fifteen  minutes  and  were  then  transferred 
to  normal  sea-water.     Practically  all  the  eggs  that  had  been 

1  Loeb,  "The  Comparative  Efficiency  of  Weak  and  Strong  Bases  in  Artificial 
Parthenogenesis,"  Jour.  Exper.  Zool.,  Xlll,  577,  1912. 


Activation  of  the  Egg  by  Bases  149 


in  0.3  c.c.  N/10  HN4OH  developed  into  larvae,  while  only  very 
few  eggs  developed  that  had  been  treated  with  the  two  strong 
bases.  In  order  to  cause  artificial  parthenogenesis  with  NaOH 
or  tetraethylammoniumhydroxide,  a  longer  exposure  is  neces- 
sary than  with  the  weak  base  HN4OH.  This  is  in  analogy  with 
our  experience  with  acids  (see  preceding  chapter). 

Not  only  NH4OH  but  other  weak  bases  like  the  amines, 
e.g.,  butylamine  or  benzylamine,  are  effective.  It  is  of  great 
interest  that  the  w^eak  base  protamine  prepared  from  the 
sperm  of  the  salmon  is  one  of  the  most  efficient  substances  for 
the  causation  of  artificial  parthenogenesis.  These  experiments 
were  made  on  the  eggs  of  Arbacia.  An  exposure  of  the  eggs 
for  only  five  minutes  to  a  solution  of  50  c.c.  m/2  NaCl+KCl 
-f  CaCU+O.S  c.c.  of  a  N/10  solution  of  one  of  these  weak  bases 
suffices  to  induce  development  in  the  egg  of  Arhada.  If  the 
eggs  are  put  afterward  for  the  proper  time  (about  twenty  to 
twenty-five  minutes  at  26°  C.)  into  any  hypertonic  solution, 
e.g.,  50  c.c.  sea-water  +  8  c.c.  2im  NaCl;  or  into  sea-water 
whose  concentration  had  been  raised  to  the  same  degree  by 
evaporation;  or  into  a  mixture  of  10  c.c.  sea-w^ater+  40  c.c.  m 
solution  of  cane  sugar,  a  certain  percentage  will  develop  into 
plutei  after  they  are  transferred  into  sea-water. 

These  results  were  confirmed  in  experiments  on  an  annelid 
(Polynoe)  of  the  Pacific  coast.  It  was  found  that  the  weak 
bases  like  amines  (methyl-,  ethyl-,  butyl-,  and  benzylamine 
were  tried)  and  NH4OH  caused  artificial  parthenogenesis  much 
more  quickly  than  the  strong  bases  NaOH  or  tetraethylam- 
moniumhydroxide.    The    amines    were    more    effective    than 

NH4OH. 

The  reason  for  this  paradoxical  behavior  of  the  bases  is  the 
same  as  for  that  of  the  acids;  the  weaker  bases  are  more  efficient 
than  the  stronger  for  the  reason  that  the  weaker  bases  diffuse 
rapidly  into  the  egg  while  the  strong  bases  do  not  diffuse  at  all 
into  the  egg  or  only  to  a  very  slight  extent.     The  direct  proof 


150     Artificial  Parthenogenesis  and  Fertilization 


of  this  statement  has  been  furnished  by  eggs  stained  red  with 
neutral  red.  Warburg  has  shown  that  if  a  small  amount  of 
NH4OH  is  added  to  the  solution  the  eggs  turn  3'ellow,  while 
when  NaOH  is  added  they  remain  red.^  Harvey  has  shown  that 
not  only  the  egg  cells  but  cells  in  general  behave  this  way, 
and  that  the  cells  are  impermeable  for  all  strong  bases  (NaOH, 
KOH,  tetraethylammoniumhydroxide)  while  they  are  perme- 
able for  weak  bases  like  NH4OH  or  the  amines.^  The  bases 
as  well  as  the  acids  must  be  able  to  diffuse  into  the  egg  in  order 
to  cause  artificial  parthenogenesis.^ 

3.  When  a  trace  of  KCN  is  added  to  the  alkaline  solution 
its  effect  is  diminished  and  the  eggs  must  either  remain  longer 
in  the  alkaline  solution  or  longer  in  the  hypertonic  solution 
in  order  to  produce  larvae.  In  former  experiments  on  the 
unfertiUzed  eggs  of  Strongylocentrotus  the  writer  could  show  that 
the  parthenogenetic  effects  (as  well  as  the  destructive  effects)  of 
NaOH  can  be  inhibited  if  the  solution  is  deprived  of  ox>'gen.^ 

4.  If  the  eggs  of  Arbacia  are  treated  with  alkali  alone, 
without  being  afterward  submitted  to  a  treatment  with  a  hyper- 
tonic solution,  they  may  form  fine  gelatinous  surface  films  (m.em- 
branes)  and  begin  to  develop,  but  they  will  then  disintegrate. 

To  50  c.c.  m/2  (NaCl+KCl-j-CaCla)  were  added  0.3  c.c. 
N/10  NH4OH,  0.3  c.c.  N/10  NaOH,  and  0.3  c.c.  N/10  tetra- 
ethylammoniumhydroxide respectively.  Unfertilized  eggs  of 
Arbacia  were  put  into  these  three  solutions  for  forty-two  min- 
utes and  then  transferred  to  normal  sea-water.  All  of  the  eggs 
that  had  been  in  the  solution  containing  the  NH4OH  segmented 
in  a  rather  amoeboid  way  into  two  or  four  cells,  after  which  the 
cells  fell  apart  and  disintegrated.     All  of  the  eggs  that  had 

1  Warburg,  Zeitschr.  f.  physiol.  Chem.,  LXVI,  305,  1910. 

2  Harvey,  Jour.  Exper.  ZooL,  X,  507,  1911. 

3  Loeb,  op.  cit. 

«  Loeb,  "Weitere  Versuche  ueber  die  Notwendigkeit  \'on  freiem  Saiierstofl 
fur  die  entwicklungserregende  Wirkung  hypertonischer  Losungen,"  I'Maer's 
Archiv,  CXVIII,  30,   1907. 


Activation  of  the  Egg  by  Bases  151 


been  in  50  c.c.  m/2  (NaCl+KCl+CaCy+O.S  c.c.  N/10 
NaOH  for  forty-two  minutes  remained  practically  intact  and 
the  same  was  true  for  the  eggs  that  had  been  in  the  tetraethyl- 
ammoniumhydroxide  for  forty-two  minutes.  In  order  to  make 
sure  that  they  did  not  only  appear  normal  but  were  normal, 
sperm  was  added  to  these  eggs  the  next  morning.  All  those 
that  had  been  in  NaOH,  and  in  tetraethylammoniumhydroxide, 
segmented  normally  and  developed  into  normal  embryos. 

In  this  experiment  part  of  the  eggs  were  submitted  for  fifteen 
minutes  to  the  action  of  the  neutral  hypertonic  solution  after 
they  had  been  treated  with  alkali.  Those  that  had  been  in 
NH4OH  developed  into  larvae,  the  others  did  not.  It  is 
obvious  that  the  changes  leading  to  parthenogenetic  develop- 
ment are  brought  about  more  rapidly  by  NH4OH  than  by  the 
strong  bases. 

5.  We  have  seen  in  the  experiments  with  the  fertilization 
of  the  eggs  by  fatty  acid  that  the  treatment  with  the  hyper- 
tonic solution  could  either  precede  or  follow  the  treatment  Avith 
fatty  acid.  In  case  it  preceded,  the  exposure  to  the  hypertonic 
solution  had  to  be  considerably  longer  than  when  it  followed  the 
fatty  acid  treatment.  The  same  is  true  for  the  fertilization 
by  bases.  If  the  eggs  of  purpuratus  are  submitted  to  the  hyper- 
tonic solution  first,  they  must  remain  much  longer  in  the  hypei'- 
tonic  solution  than  if  they  are  put  into  it  after  having  undergone 
a  treatment  with  alkali.  This  is  due  to  the  difference  in  the  rate 
of  oxidations  in  the  egg  before  and  after  membrane  formation. 
When  the  rate  of  oxidation  is  higher  in  the  egg  the  exposure  to 
the  hypertonic  solution  may  be  shorter. 

It  may  be  stated  incidentally  that  artificial  parthenogenesis 
by  bases  is  less  satisfactory  in  S.  purpuratus  than  in  Arhacia 
since  only  the  eggs  of  perhaps  one-third  of  the  females  of 
purpuratus  develop  into  larvae  through  the  influence  of  bases. 
Where  the  method  is  successful  it  can  be  shown  that  NH4OH, 
and  to  some  degree  the  amines,  are  more  effective  than  NaOH. 


152     Artificial  Parthenogenesis  and  Fertilization 


6.  The  inhibitive  effect  of  KCN  and  lack  of  oxygen  upon  the 
activation  of  the  unfertilized  egg  by  bases  suggested  an  investi- 
gation of  the  influence  of  the  various  bases  upon  the  rate  of 
oxidations  in  the  unfertilized  egg,  to  find  out  whether  the  weaker 
bases  raised  the  rate  of  oxidations  more  than  the  stronger  ones.^ 

The  experiments  were  carried  out  on  the  unfertilized  egg 
of  S.  piirpuratus.  The  oxygen  content  was  determined  ac- 
cording to  Winkler's  method.  The  experiments  were  made 
in  a  half  grammolecular  mixture  of  NaCl+KCl+CaCla  in  that 
proportion  in  which  these  three  salts  are  contained  in  the  sea- 
water. 

We  first  give  the  results  of  a  series  of  experiments  in  w^hich 
the  relative  influence  of  various  bases   was   compared.     The 


TABLE  XXVI 


Number 

of 
Experi- 
ment 


I.. 
II.. 


III. 
IV. 


V. 

VI. 

VII. 

VIII. 


Nature  of  the  Solution 


/Neutral 

\50  c.c.  neutral +0.3  c.c.  N/10  NaOH. .  . 

/Neutral 

\50  c.c.   neutral +0.3   c.c.   N/10   tetra- 
ethylammoniumhydroxide 


/Neutral 

150  c.c.  neutral+0.3  c.c.  N/10  NH^OH. 

/Neutral 

\50    c.c.     neutral+0.3     c.c.    N/10   tri- 
methylamine 


mgm.  of 

Oxygen 

Consumed 


/Neutral 

\50  c.c.  neutral+0.3  c.c.  N/10  methyl- 
amine   

/Neutral 

\50  c.c.  neutral+0.3  c.c.    N/10    ethyl 

amine 

Neutral 

50  c.c.    neutral+0.3    c.c.    N/10  butyl 
amine 

50  c.c.   neutral+0.3   c.c.  N/IO  benzyl- 
amine 


0.28 
0.40 
0.15 

0.22 


0.30 
0.81 
0.40 

1.19 


0.25 

1.18 

0.28 

1.35 
0.32 

1.23 
0.22 

1.30 


Acceleration 

of  Rate  of 

Oxidations 

by  the  Base 


1.43 
1.50 


2.70 
3.00 


4.70 
4.80 
3.80 
5.90 


1  Loeb  and  W^asteneys,  Jour.  Biol.  Chem.,  XIV,  35.5,  1913. 


Activation  of  the  Egg  by  Bases 


153 


time  of  exposure  was  one  hour  and  twenty-five  minutes;  the 
temperature  18°  C.  The  concentration  of  the  bases  chosen 
was  that  found  most  effective  in  the  writer's  previous  experi- 
ments on  artificial  parthenogenesis.  The  oxygen  consumption 
was  first  measured  in  a  neutral  solution  and  then  for  the  same 
eggs  in  the  alkaline  solution  in  which  0 . 3  c.c.  N/10  of  the  various 
bases  was  added  to  50  c.c.  of  the  solution. 

This  influence  of  the  various  bases  upon  the  rate  of  oxida- 
tion in  the  eggs  of  purpuratus  runs  parallel  to  their  relative 
efficiency  in  causing  development  in  Polynoe.  To  induce 
artificial  parthenogenesis  in  the  eggs  of  Polynoe  the  simple 
amines  were  found  to  be  most  eflftcient,  next  came  NH4OH  and 
trimethylamine,  and  finally  the  strong  bases  NaOH  and  tetra- 
ethylammoniumhydroxide.  The  amines  seem  to  hurt  the  eggs 
of  purpuratus  more  than  those  of  Polynoe. 

Incidentally  it  may  be  stated  that  NaHCOg  does  not 
accelerate  the  rate  of  oxidations  in  the  unfertilized  egg,  nor 
does  it  cause  artificial  parthenogenesis. 

7.  We  compared  next  the  relative  effect  of  various  concen- 
trations of  NaOH  and  NH4OH  upon  the  rate  of  oxidations  in 
the  unfertilized  sea-urchin  egg,  during  one  hour.  We  will 
state  only  the  coeflficient  of  the  rate  of  oxidation  in  the  various 
solutions  calling  the  rate  in  the  neutral  solution  1.00. 

TABLE  XXVII 


Amount  of  Base  Added  to 

50  c.c.  m/2  (NaCl+KCl+ 

CaCL) 


Coefficient  of  Accel- 
eration of  Oxid.i- 
tions  in 


154     Artificial  Parthenogenesis  and  Fertilization 


The  reader  will  notice  the  striking  difference  in  the  behavior 
of  NH4OH  and  NaOH.  Low  concentrations  of  NH4OH  (0.5 
CO.  per  50  c.c.  solution)  raise  the  rate  of  oxidations  in  the 
fertilized  egg  almost  to  the  maximal  height  and  a  further  rise 
in  the  concentration  has  only  a  slight  effect  upon  the  rate  of 
oxidation.  Low  concentrations  of  NaOH  raise  the  rate  of  oxi- 
dation only  a  little,  and  the  efficiency  of  NaOH  rises  steadily 
with  an  increase  in  its  concentration.  We  could  not  go  beyond 
the  concentrations  used  in  this  experiment,  since  the  addition 
of  3  c.c.  N/10  NaOH  to  50  c.c.  m/2  NaCl+KCl+CaCU  leads 
already  to  a  cytolysis  of  the  eggs. 

It  is  also  of  interest  to  point  out  that  in  the  eggs  of  S.  pur- 
puratus  fertilization  by  sperm  raises  the  rate  of  oxidation  to 
about  five  or  six  times  the  amount  of  that  in  the  unfertilized 
eggs.  This  seems  to  indicate  that  with  NH4OH  it  is  not  pos- 
sible to  raise  the  rate  of  oxidations  in  the  unfertilized  egg 
beyond  the  limit  to  which  it  can  be  raised  by  the  fertilization 
with  sperm.  It  is  not  possible  to  decide  whether  the  same 
holds  true  for  NaOH. 

The  fact  that  a  base  reaches  its  maximum  effect  at  so  low 
a  concentration  is  not  confined  to  NH4OH  but  is  also  shared 
by  the  amines  as  the  following  table  shov/s.  NH4OH  and 
ethylamine  were  compared. 

TABLE  XXVIII 


Amount  of  Base  Added  to 
50  c.c.  m/2  (NaCl+KCl+ 

Coefficient  of  Acceij- 
eration  of  oxida- 
TIONS IN 

CaCh) 

NH4OH 

E  thylamine 

0  1  c  c   N/10 

1.9 
2.9 
3.4 
3.9 

1.4 

0  2  c  c   N/10 

3.0 

0  4  c  c  N/10 

4.3 

0  8  0  0  N/10        

4.2 

Ethylamine  reaches  its  maximal  efficiency  at  the  concentra- 
tion of  0.4  c.c.  base  to  50  c.c.  of  the  neutral  liquid;    and  for 


Activation  of  the  Egg  by  Bases 


155 


NH4OH  the  limit  is  nearly  at  the  same  point  as  in  our  previous 
experiment. 

8.  It  seemed  natural  to  connect  this  difference  in  the 
behavior  of  NaOH  and  NH4OH  with  the  difference  in  the  rate 
of  their  diffusion  into  the  unfertilized  egg.  If  the  rate  of 
diffusion  of  NaOH  is  extremely  slow,  and  that  of  NH4OH  fast,  it 
is  natural  that  the  maximal  rate  of  oxidation  should  be  reached 
with  a  lower  concentration  of  NH4OH  than  of  NaOH.  We 
determined  the  consumption  of  oxygen  for  the  same  lot  of 
eggs  for  eight  consecutive  hours  in  50  c.c.  sea-water -f  1.0  c.c. 
N/10  NaOH.     Table  XXIX  gives  the  result.     This  shows  that 


TABLE  XXIX 

Consumption  of  Oxygen  by  Unfertilized  Eggs  at  18**  in  50  c.c. 
Normal  Sea-Water+1.0  c.c.  N/10  NaOH 


Oxygen 
Consumed 

Coefficient 
of  Oxidation 

1st  hour 

TTigm. 

0.24 

0.38 

0.45 

0.50 

0.58 

0.72 

0.92 

0.95 

1  00 

2d  hour 

1  57 

3d  hour 

1  87 

4th  hour 

2  08 

5th  hour 

2  42 

6th  hour 

3  00 

7th  hour . 

3  84 

8th  hour 

3  96 

the  longer  the  NaOH  acts  upon  the  egg  the  higher  the  amount 
of  ox^^gen  becomes  which  is  consumed  per  hour.  This  would 
agree  with  the  assumption  that  the  NaOH  diffuses  slowly  into 
the  egg  and  that  the  increase  in  the  rate  of  oxidations  in  the 
unfertilized  egg  is  determined  by  the  amount  of  base  which 
has  diffused  into  the  egg. 

It  was  expected  that  since  NH4OH  is  very  soluble  in  the 
egg,  i.e.,  diffuses  rapidly  into  it,  its  maximum  effect  would  be 
reached  during  the  first  hour.  This  was  found  to  be  true,  as 
Table  XXX  shows. 


156     Artificial  Parthenogenesis  and  Fertilization 


We  intend  to  investigate  whether  these  effects  of  bases  upon 
the  rate  of  oxidations  in  the  unfertihzed  eggs  are  irreversible, 
i.e.,  will  continue  if  the  eggs  are  put  into  normal  sea-water  after 
the  treatment  with  alkali.  But  we  have  an  experiment  which 
possibly  serves  the  same  purpose.  We  measured  the  amount 
of  ox>'gen  consumed  in  one  hour  in  the  eggs  mentioned  in 
Table  XXX  in  the  same  solution  sixteen  and  twenty-four  hours 

TABLE  XXX 
Consumption  of  Oxygen  by  Unfertilized  Eggs  at  18"  in  50  c.c. 
Normal  Sea-Water+0.8  c.c.  N/10  NH4OH 


Oxygen 
Consumed 

Coeflficient 
of  Oxidation 

mgm. 

Normal  sea-water 

0.15 

1.0 

50  c.c.  sea-water +0.8  c.c 

N/10  NH4OH; 

Ist  hr. 

0.99 

6.7 

50  c.c.  sea-water+0.8  c.c. 

N/10  NH4OH; 

2dhr.. 

1.03 

6.9 

50  c.c.  sea-water+0.8  c.c. 

N/10  NH4OH; 

3dhr.. 

0.87 

5.8 

50  c.c.  sea-water+0.8  c.c. 

N/10  NH4OH; 

4th  hr. 

0.86 

5.7 

50  c.c.  sea-water+0.8  c.c. 

N/10  NH4OH; 

5th  hr . 

0.83 

5.5 

after  the  experiment.  In  the  meantime  the  eggs  had  been  kept 
at  a  low  temperature  in  normal  sea-water.  The  rate  of  oxida- 
tions after  sixteen  or  twenty-four  hours  was  practically  the 
same  as  in  the  second  hour.  This  agrees  with  the  assumption 
that  these  bases  bring  about  the  modification  of  the  cortical 
layer  of  the  egg,  after  which  the  rate  of  oxidation  in  the  egg  is 
raised  permanently. 

These  experiments  prove  two  facts,  first,  that  the  weaker 
bases  increase  the  rate  of  oxidations  in  the  unfertilized  egg  more 
than  the  stronger  bases;  and,  second,  that  this  difference  is  due 
to  the  fact  that  the  weaker  bases  diffuse  more  rapidly  into  the 
egg  than  the  strong  bases. 

The  connection  between  the  oxidative  action  of  bases  and 
artificial  parthenogenesis  lies  in  the  fact  that  the  essential 
factor  in  artificial  parthenogenesis  is  an  alteration  of  the 
surface  or  cortical  layer  of  the  egg  which  results  in  a  membrane 


Activation  of  the  Egg  by  Bases  157 


formation.  We  shall  see  later  that  bases  cause  the  swelling 
and  liquefaction  of  the  gelatinous  mass  (the  so-called  chorion) 
which  surrounds  the  immature  egg  of  a  mollusc,  Lottia,  and  that 
this  action  of  bases  is  inhibited  by  lack  of  oxygen  and  by  the 
addition  of  KCN.^  This  year  the  author  convinced  himself 
that  weak  bases  like  the  amines  and  NH4OH  bring  about  the 
dissolution  of  the  chorion  of  Lottia  much  more  rapidly  than 
the  strong  bases  NaOH  and  tetraethylammoniumhydroxide. 
It  is  possible  that  the  induction  of  artificial  parthenogenesis 
in  the  sea-urchin  egg  by  bases  depends  upon  the  occurrence  of 
a  similar  process  in  the  cortical  layer  of  this  egg.  We  may 
imagine  that  the  bases  act  by  accelerating  the  rate  of  oxida- 
tion of  a  substance  (existing  in  the  cortical  layer  of  the  eggs) 
whereby  the  membrane  formation  and  consequently  the  devel- 
opment of  the  egg  is  induced. 

1  Loeb,  University  of  California  Publications,  Physiology,  III,  1,  1905. 


XVI 

ANALYSIS  OF  THE  ORIGINAL  METHOD  OF  PRODUCING 
ARTIFICIAL  PARTHENOGENESIS  BY  HYPERTONIC 

SOLUTIONS  ALONE 

1.  We  are  now  able  to  undertake  the  analysis  of  the  purely 
osmotic  method  of  artificial  parthenogenesis  by  which  the 
writer  first  obtained  parthenogenetic  larvae  of  the  sea-urchin 
(see  chapter  vii).  The  reader  will  notice  that  this  method  ap- 
parently contradicts  the  statement  that  artificial  partheno- 
genesis is  due  to  the  influences  of  two  agencies,  one  of  which 
causes  the  membrane  formation,  the  second  the  correcting 
effect,  which  protects  the  egg  against  the  disintegration  in- 
duced by  the  artificial  membrane  formation.  The  apparent 
contradiction  is  abolished  by  the  realization  of  the  fact  that 
in  this  method  the  hypertonic  solution  acts  simultaneously  in 
two  capacities:  first,  as  a  cytolytic  agency  causing  a  change 
in  the  cortical  layer  (the  formation  of  a  gelatinous  film),  and 
second,  as  a  corrective  agency.  This  latter  effect  is  sufficiently 
intelligible  from  what  has  been  said  before.  The  cytolytic 
effect  of  a  hypertonic  solution,  however,  needs  to  be  demon- 
strated, and,  moreover,  proof  must  be  furnished  that  the  two 
agencies  active  in  artificial  parthenogenesis  may  act  simultane- 
ously instead  of  in  succession. 

It  must  first  be  stated  that  this  method  also  leads  to  a  kind 
of  stunted  membrane  formation;  only  such  eggs  can  be  caused 
to  develop  by  this  method  which  form  a  membrane. 

But  the  hypertonic  solution  is  not  a  reliable  agency  for  the 
causation  of  membrane  formation,  and  this  is  the  reason  that 
this  original,  purely  osmotic  method  of  artificial  parthenogenesis 
gives  such  poor  results  with  the  eggs  of  S.  purpuratus  and 
not  very  good  results  with  the  eggs  of  Arbacia.     The  direct 

159 


160     Artificial  Parthenogenesis  and  Fertilization 

membrane  formation  by  butyric   acid  is  much  more  reliable 
than  that  by  the  hj'pertonic  solution. 

The  membrane-forming  action  of  a  hypertonic  solution  is 
similar  to  that  of  a  base,  and  this  can  be  denjonstrated  in  the 
liquefaction  of  the  chorion  of  the  egg  of  Lottia  (a  mollusc). 
We  have  mentioned  that  this  chorion  can  be  caused  to  swell 
and  undergo  liquefaction  by  bases.  The  writer  found  that  the 
same  can  be  accomplished  by  neutral  hypertonic  solutions.^  In 
both  cases  the  presence  of  free  oxygen  is  required.  Moreover, 
the  temperature  coefficient  for  the  dissolution  of  the  chorion,  in 
the  unfertilized  egg  of  Lottia  by  hypertonic  solutions  is  over 
2  for  10°  C. 

The  idea  that  in  the  causation  of  artificial  parthenogenesis 
by  the  original  purely  osmotic  method  the  latter  acted  in  the 
double  capacity  of  a  membrane-forming  and  corrective  agency 
can  be  supported  bj^  measurements  of  the  rate  of  oxidation  of 
unfertilized  eggs  in  these  solutions. 

According  to  our  theory  the  rise  in  the  rate  of  oxidations  in 
unfertilized  eggs  under  the  influence  of  hypertonic  solutions 
should  be  due  only  to  the  alteration  of  the  surface  of  the 
egg,  i.e.,  the  membrane  formation.  Since  this  effect  takes 
place,  if  it  takes  place  at  all,  in  the  first  two  hours,  we  should 
expect  that  if  we  leave  unfertilized  eggs  for  a  series  of  hours 
in  a  hypertonic  solution,  and  measure  the  rate  of  oxida- 
tion for  successive  hours,  the  maximum  effect  should  be  reached 
in  the  first  or  second  hour  and  that  afterward  the  hypertonic 
solution  should  cause  no  further  rise  in  the  rate  of  oxidations. 
This  actually  takes  place.  The  following  experiment  gives  the 
rate  of  oxidations  of  unfertilized  eggs  in  a  hypertonic  solution 
in  a  series  of  successive  hours. ^  The  consumption  of  oxygen 
was  measured  for  one  hour   at   18°.     Unfertilized  eggs  were 

1  Loeb,  "  On  a  Chemical  Method  by  Which  the  Eggs  of  Lottia  Can  Be  Caused 
to  Become  Mature,"  University  of  California  Publications,  Physiology,  III,  1,  1905. 

2  Loeb  and  Wasteneys,  Jour.  Biol.  Chem.,  XIV,  469,  1913. 


Original  Method  of  Artificial  Parthenogenesis     161 


put  directly  into   hypertonic   sea-water   (50    c.c.   sea-water + 
8  c.c.  2i  m  NaCl).     Temperature  18°  C. 

The  maximum  consumption  of  oxygen  was  reached  in  the 
second  hour  in  which  the  effect  of  membrane  formation  was 
ahnost  complete.     In  this  case  the  eggs  of  a  female  were  used 

TABLE  XXXI 
Unfertilized  Eggs 


Normal  sea-water .... 
Hypertonic  sea- water; 


1st  hour. 
2d  hour . 
3d  hour. 
4th  hour , 
5th  hour . 


Oxygen 
Consumed 


'     Coefficient  of 
Rate  of  Oxidation 


0.16mg. 

0.67 

0.79 

0.64 

0.56 

0.57 


1.0 
4.2 
4.9 
4.0 
3.6 
3.2 


which  w^ere  obviously  very  susceptible  to  this  treatment.  The 
effect  of  the  hypertonic  solution  upon  the  rate  of  oxidation  in 
the  unfertilized  egg  was  as  great  as  that  produced  by  artificial 
membrane  formation  through  butyric  acid  or  by  fertilization 

with  sperm. 

In  many  cases  the  treatment  of  eggs  of  S.  piirpuratus  with 
hypertonic  sea-water  leads  to  no  membrane  formation  and  no 
development,  or  only  to  the  development  of  a  limited  number 
of  eggs.  Consequently  the  rise  in  the  rate  of  oxidation  caused 
in  such  cases  should  be  smaller  than  that  caused  by  artificial 
membrane  formation  with  butyric  acid  which  usually  takes 
effect  in  practically  all  the  eggs. 

We  give  in  Table  XXXII  a  series  of  experiments  with 
hypertonic  sea-water  of  various  concentrations.  It  should  be 
remembered  that  the  addition  of  4  c.c,  or  less,  2h  m  Ringer  to 
50  c.c.  of  sea-water  was  without  effect.  The  oxygen  consump- 
tion was  first  measured  in  normal  sea-water,  then,  for  the  same 
quantities  of  eggs,  in  a  hypertonic  solution.  The  table  gives 
the  ratio  of  the  oxj^gen  consumption  in  hypertonic  to  that 
in  normal  sea-water. 


162     Artificial  Parthenogenesis  and  Fertilization 

In  this  case  the  rise  in  the  rate  of  oxidations  is  less  than 
that  ordinarily  caused  by  membrane  formation  with  butyric 
acid  in  the  same  eggs. 

TABLE  XXXII 

Unfertilized  Eggs  of  S.  purpuratus 


Coefficient  of 

Oxygen 

Consumption 

50  c.c. 

sea-water  +  4  c.c. 

21  m  NaCl+KCl+CaCL 

1.4 

50  c.c. 

sea-water  +  6  c.c. 

21  m  NaCl+KCl+CaCl. 

1.9 

50  c.c. 

sea- water  +  8  c.c. 

2h  m  NaCl+KCl+CaCl. 

2.6 

50  c.c. 

sea- water  +  9  c.c. 

2h  m  NaCl+KCl+CaCL 

2.6 

50  c.c. 

sea-water +  12  e.c. 

2h  m  NaCl+KCl+CaCL 

2.3 

50  c.c. 

sea-water +  16  c.c. 

2^  m  NaCI+KCl+CaCl. 

2.6 

2.  The  hAT^ertonic  solutions  are  much  more  effective  in 
causing  artificial  parthenogenesis  if  some  alkali  is  added.  In 
one  experiment  0,  0.5,  1.0,  1.5,  and  2.0  c.c.  N/10  NaOH  were 
added  each  to  50  c.c.  of  sea-water +10  c.c.  2|  m  NaCl  solution. 
The  unfertilized  eggs  of  one  specimen  of  S.  purpuratus  were 
divided  among  these  solutions  and  portions  of  the  eggs  trans- 
ferred to  normal  sea-water  after  60,  90,  120,  150,  and  240 
minutes.  The  temperature  of  the  hypertonic  sea-water  was 
13 . 5°  C.  Only  the  two  of  these  solutions  with  the  highest 
concentration  of  NaOH  (1.5  and  2.0  c.c.  N/10  NaOH  to 
50  c.c.  of  the  hypertonic  sea-water)  caused  the  eggs  to 
develop  into  larvae  (time  of  exposure  1^  hours).  Some  of 
these  larvae  reached  the  pluteus  stage  and  swam  at  the  sur- 
face of  the  dish.  Often,  however,  the  addition  of  quite  a  small 
amount  of  NaOH  was  enough  to  cause  development  and  for 
the  eggs  of  some  females  the  concentration  of  HO  ions  pres- 
ent in  the  sea-water  is  sufficient.  We  are  dealing  here  with 
differences  in  the  eggs  of  various  females.  It  was  found  in 
general  that  a  neutral  hypertonic  solution  was  not  able  to  induce 
artificial  parthenogenesis  in  the  eggs  of  S.  purpuratus.  Even 
the  maximal  permissible  increase   of  the  osmotic  pressure  is 


Original  Method  of  Artificial  Parthenogenesis     1(33 

generally,  though  possibly  not  always,  unable  to  cause  the 
unfertilized  eggs  of  S.  purpuratus  to  develop  into  larvae,  no 
matter  how  long  the  egg  is  exposed  to  such  a  solution.  For  such 
experiments  the  alkaline  sea-water  cannot  be  used;  hence  the 
writer  chose  as  a  neutral  solution  a  neutral  mixture  of  100 
c.c.  NaCl,  2.2  c.c.  KCl,  1.5  c.c.  CaCla,  and  11.6  c.c.  MgClj, 
all  the  solutions  being  half  grammolecular. 

We  call  such  a  van't  Hoff  solution  neutral  when  it 
is  colored  red  by  neutral  red,  but  turns  to  orange  for  a  few 
minutes  on  the  addition  of  only  0.1  c.c.  of  N/100  NaOH, 
afterward  becoming  red  again.  The  concentration  of  the 
hydroxylions  in  such  a  solution  lies  below  10"^  normal,  but  so 
close  to  this  value  that  the  slightest  addition  of  alkali  brings 
up  the  concentration  of  the  HO  ions  to  this  amount ;  however, 
owing  to  the  diffusion  of  the  CO2  of  the  air  into  the  solution, 
the  concentration  of  the  HO  ions  soon  sinks  again  below  the 
limit  of  10"®  n. 

We  shall  now  illustrate  by  a  few  examples  the  fact  that 
even  the  greatest  increase  in  osmotic  pressure  will  as  a  rule  not 
,  effect  the  transformation  of  eggs  of  S.  purpuratus  into  larvae  in 
neutral  solution.  To  50  c.c.  of  a  neutral  van't  Hoff  solution, 
8,  12,  16,  24,  and  32  c.c.  of  2|  m  KCl  solution  were  added 
respectively.  The  eggs  of  one  female  were  divided  among  these 
solutions  after  being  washed  twice  in  the  van't  Hoff  solution 
to  free  them  from  any  trace  of  sea-water.  This  precaution 
is  essential  in  such  investigations,  and  has  been  employed  in  all 
our  experiments.  The  temperature  of  the  hypertonic  solution 
was  13°  C.  A  portion  of  the  eggs  was  transferred  from  each 
solution  to  normal  sea-water  after  25,  45,  75,  105,  145,  185,  and 
220  minutes.  Not  a  single  egg  developed  into  a  larva.  With 
the  addition  of  32  c.c.  of  2i  m  KCl  to  50  c.c.  of  the  van't  Hoff 
solution  the  limit  of  the  permissible  osmotic  pressure  is  reached, 
since  at  higher  osmotic  pressures  the  eggs  are  at  once  cytolyzed.' 

iLoeb,  Pfluger's    Archiv,  CIII,   257,    1904;     Untersuchungen   ueber  die   kunst- 
liche  Parthenogenese,  pp.   288  flf. 


164    Artificial  Parthenogenesis  and  Fertilization 

The  following  experiment,  in  whicli  sea-water  was  used  instead 
of  the  van't  Hoff  solution,  is  also  instructive.  Eggs  were  used 
for  which  the  concentration  of  the  hydroxy  lions  in  sea-water 
was  not  high  enough  to  permit  of  their  development.  To  50  c.c. 
of  sea-water  4,  8,  12,  16,  24,  and  32  c.c.  2^  m  NaCl  solution 
were  added,  and  some  of  the  eggs  of  one  female  were  divided 
among  these  solutions.  After  20,  40,  70,  100,  135,  210,  273,  and 
346  minutes  portions  of  the  eggs  were  replaced  in  normal  sea- 
water.  In  no  case  did  even  a  single  egg  develop  to  a  larva. 
Many  eggs  started  cleavage;  but  this  soon  ceased,  a  fact  to 
which  we  shall  return  later.  It  might  have  been  supposed  that 
here  one  was  dealing  with  eggs  which  could  not  be  caused  to 
develop  in  any  way  at  all  by  the  osmotic  effect.  However,  it 
was  possible  to  show  that  it  depended  upon  too  low  a  concentra- 
tion of  hydroxylions.  Thus  a  control  experiment  was  per- 
formed, in  which  0,  0. 1,  0.2,  0.4,  and  0.8  c.c.  of  N/10  NaOH 
were  added  to  50  c.c.  of  van't  Hoff's  solution -f  16  c.c.  of  2J  m 
NaCl  and  some  of  the  eggs  of  the  same  female  were  divided 
among  these  solutions.  After  30,  60,  90,  120,  and  210  min- 
utes portions  of  these  eggs  were  transferred  to  normal  sea- 
water.  Of  the  eggs  treated  with  neutral  van't  Hoff's  solution 
not  a  single  one  developed  into  a  larva,  while  the  eggs  treated 
for  90  to  120  minutes  with  alkaline  solution  produced  larvae; 
the  larvae  were  most  numerous  in  that  portion  of  the  eggs 
which  had  been  for  90  minutes  in  the  solution  with  0.8  c.c. 
N/10  NaOH.  Of  this  portion  80  per  cent  of  the  eggs  devel- 
oped into  larvae. 

On  the  other  hand  it  was  possible  to  show  that  in  hyper- 
alkaline  solutions  a  relatively  small  increase  of  the  osmotic 
pressure  was  sufficient  to  cause  the  formation  of  larvae  in 
unfertilized  eggs,  and  that  any  further  increase  of  osmotic  pres- 
sure only  diminished  the  time  of  exposure  to  the  solution  neces- 
sary for  the  formation  of  larvae.  The  following  experiment  will 
serve  as  an  example.     To  50  c.c.  of  the  van't  Hoff  solution -f- 


Original  Method  of  Artificial  Parthenogenesis    105 

2.0  c.c.  N/10  NaOH,  0,  2,  4,  8,  and  16  c.c.  of  2|  m  KCl  were 
added.  Unfertilized  eggs  were  divided  among  these  five  solu- 
tions and  portions  of  the  eggs  transferred  to  normal  sea-water 
after  45,  64,  89,  116,  and  144  minutes.  The  results  are  sum- 
marized in  Table  XXXIII.  The  increase  of  osmotic  pressure 
is  given  in  the  table  in  round  numbers  as  a  percentage  of  the 
pressure  of  the  half  grammolecular  solution. 


TABLE  XXXIII 


Time  of 

Increase  of  the  Osmotic  Pressure  of  the  Solution 

Exposure 

0  Per  Cent 

16  Per  Cent 

30  Per  Cent 

55  Percent 

87  Per  Cent 

45  minutes.  .  .  . 

64  minutes .... 

89  minutes .... 

116  minutes.  .  .  . 
144  minutes .... 

0 

0 

0 

0 
0 

0 

0 

0 

0 
0 

0 

0 

numerous 
larvae 

0 

numerous 
larvae 

numerous 
larvae 

Further  experiments  on  the  eggs  of  S.  purpuratus  showed 
that  the  results  remain  the  same  if  the  two  agencies,  the  base 
and  the  neutral  hypertonic  solution,  are  applied  in  succession 
instead  of  simultaneously.  When  the  treatment  of  these  eggs 
with  the  alkaline  solution  (added  to  a  neutral  isotonic  solution) 
preceded  their  treatment  with  a  neutral  hypertonic  solution, 
it  could  be  seen  that  the  former  agency  acted  mainly  as  the 
membrane-forming  agency,  w4iile  the  hypertonic  solution  acted 
as  the  corrective  agency. 

For  the  egg  of  Arbacia  a  neutral  hypertonic  solution  suffices 
to  call  forth  a  normal  development. 

3.  Why  should  the  addition  of  alkali  to  the  hypertonic 
solution  increase  its  efficiency?  The  answer  is  that  the  ad- 
dition of  alkali  increases  the  membrane-forming  effect  of  the 
hypertonic  solution.     In  S.  purpuratus  the  hypertonic  solution 


166     Artificial  Parthenogenesis  and  Fertilization 


alone  is  not  able  to  induce  the  (stunted)  membrane  formation 
in  the  eggs,  but  the  addition  of  alkali  increases  the  membrane- 
forming  power.  This  can  be  proved  by  the  fact  that  the  ad- 
dition of  alkaU  to  the  hypertonic  solution  causes  a  rise  in  the 
rate  of  the  oxidations  of  almost  the  same  order  as  the  addition 
of  the  same  amount  of  base  to  an  isotonic  sohition  (Table 
XXXIV). 

TABLE  XXXIV 


Number  of 
Experiment 


II 


III 


Unfertilized  Eggs  (without  Membranes)  in 


Oxj-gen 
Consumed 


Xonnal  sea-water 

50   c.c.  hypertonic  sea-water +  1   c.c. 

X/10  NH4OH 

50  c.c.  normal  sea-water +  1  c.c.  N/10 

NH4OH 

Noi-mal  sea- water 

50  c.c.  hypertonic   sea-water +  1    c.c. 

N/10  benzylamine 

50  c.c.  normal  sea-water +  1  c.c.  N/10 

benzylamine   

Normal  sea- water 

50  c.c.  hypertonic  sea-water  +  1   c.c. 

N/10  butylamine 

50  c.c.  normal  sea-water+1  c.c.  N/10 

butylamine   


mgm. 
0.22 

1.20 

0.88 
0.37 

1.89 

1.75 
0.36 


1.73 


1.67 


CoeflBcient 

of  Rate  of 

Oxidations 


1.00 

5.40 

4.00 
1.00 

5.10 

4.70 
1.00 

4.80 

4.60 


It  is  obvious  that  the  weak  base  alone  raises  the  rate  of 
oxidations  practically  to  the  same  height  as  the  combination 
of  base  and  hypertonic  sea-water.  The  whole  rise  was  due  in 
both  cases  to  the  membrane-forming  effect  for  which  the  weak 
base  was  sufficient. 

In  the  case  of  a  strong  base,  the  result  may  be  different, 
since  in  purpuratus  neither  the  base  nor  the  hypertonic  solu- 
tion alone  may  cause  membrane  formation  (or  the  change  in 
the  cortical  layer  of  the  egg)  necessary  for  development.  The 
following  may  serve  as  an  example  (Table  XXXV).  Duration 
of  experiment  one  and  one-half  hours;  temperature  18°  C. 


Original  Method  of  Artificial  Parthenogenesis    167 


In  this  case  the  NaOH  had  Uttle  effect  and  hence  the 
hypertonicit}^  caused  a  noticeable  increase  in  the  rate  since  it 
probably  increased  the  number  of  eggs  in  which  the  process  of 
membrane  formation  was  started. 

TABLE  XXXV 


Unfertilized  Eggs  (without  Membrane 
Formation)  in 

Oxygen 
Consumed 

Coefficient 
of  Rate  of 
Oxidations 

Normal  sea- water 

mgm. 
0.41 
0.81 
0.46 

1  00 

50  c.c.  hypertonic  sea-water  +  1  c.c.  N/10  NaOH 
50  c.c.  normal  sea-water  +  1  c.c.  N/10  NaOH  .... 

2.00 
1.20 

4.  It  is  necessary  for  the  developmental  effect  that  the 
hypertonic  solution  contain  free  oxygen.  If  the  ox^^gen  is 
driven  out  of  a  hypertonic  solution  sufficiently  thoroughly, 
this  solution  can  no  longer  cause  the  unfertilized  eggs  of  the 
sea-urchin  to  develop.  The  following  experiment  on  the  eggs 
of  S.  purpuratus  will  serve  as  an  example.  The  air  was  driven 
out  of  a  series  of  flasks  each  of  which  contained  50  c.c. 
of  sea-water +8  c.c.  2 J  m  NaCl/  by  passing  chemically 
pure  hydrogen  through  them  for  several  hours,  and  then 
a  pipette  full  of  sea-urchin  eggs  was  introduced  into  each 
of  them.  This  was  effected  with  the  aid  of  an  assistant  and  in 
such  a  way  that  the  stopper  of  the  flask  was  lifted  to  one  side 
over  the  rim  for  only  one  or  two  seconds  and  during  this  time 
the  pipette  that  had  been  held  in  readiness  was  emptied  into 
the  flask.  The  current  of  hydrogen  was  not  interrupted  and 
proceeded  throughout  the  whole  experiment.  Some  of  the  eggs 
were  placed  as  a  control  in  50  c.c.  of  sea-water +8  c.c.  of  2^  m 
NaCl  which  remained  in  contact  with  air.  After  128  and  180 
minutes  samples  of  the  eggs  w^ere  replaced  in  normal  sea-water. 
The  control  eggs  which  had  been  in  the  ox^'genated  sea-water 
and  were  transferred  to  normal  sea-water  after  128  minutes 
developed  practically  all  into  larvae;  of  the  eggs  transferred  to 

1  This  hypertonic  solution  was  slightly  alkaline. 


168    Artificial  Parthenogenesis  and  Fertilization 

normal  sea-water  after  180  minutes  practically  all  disintegrated 
into  droplets  and  onlj'  a  few  developed.  Not  a  single  one  of  the 
eggs  which  had  been  in  the  hj-pertonic  solution  in  an  atmosphere 
of  h^'drogen  developed  when  transferred  to  normal  sea-water 
after  128  and  180  minutes.  The  eggs  appeared  quite  normal 
and  that  they  were  really  so  is  shown  by  the  fact  that  they 
developed  normally  upon  the  addition  of  sperm. 

In  these  experiments,  of  course,  not  all  the  ox^'gen  had  been 
expelled  from  the  hj^pertonic  solution,  but  the  pressure  of  the 
oxygen  had  been  depressed  below  the  minimum  necessary  for 
the  developmental  effect  of  the  hypertonic  solution. 

We  will  mention  one  further  experiment.  Unfertilized  eggs 
of  a  female  were  divided  among  five  flasks,  each  of  which  con- 
tained 50  c.c.  of  sea-water +8  c.c.  2 J  m  NaCl.  One  flask  was 
left  open,  i.e.,  exposed  to  the  air;  the  others,  out  of  which  the 
air  had  been  driven  by  passing  hydrogen  through  them  for  two 
hours,  remained  connected  to  the  stream  of  hydrogen.  After 
2,  3,  4|,  and  5|  hours,  one  of  the  flasks  was  disconnected  from 
the  hydrogen  apparatus,  and  the  eggs  contained  in  it  were 
transferred  to  normal  aerated  sea-water.  They  were  abso- 
luteh^  unaltered  and  chd  not  divide  or  develop.  On  the 
addition  of  sperm^  they  all  segmented  and  developed  normally, 
thus  showing  that  they  were  really,  not  only  apparently, 
unchanged.  Simultaneously  a  sample  of  the  eggs  from  the 
aerated  hypertonic  sea-water  was  also  immediately  transferred 
to  normal  sea-water.  The  eggs  transferred  from  this  hyper- 
tonic to  normal  sea-water  after  two  hours  yielded  a  good  number 
of  normal  blastulae.  Only  about  1  per  cent  of  the  eggs,  trans- 
ferred from  the  aerated  hypertonic  to  normal  sea-water  after 
three  hours,  developed;  the  rest  disintegrated  into  small  drops. 
Those  removed  after  4 J  and  5 J  hours  all  went  to  pieces  in  the 
same   way.     We   can   now   understand   another   phenomenon 

1  The  sperm  should  not  be  added  until  about  an  hour  after  the  eggs  are 
replaced  In  oxygenated  sea-water;  otherwise  the  results  are  not  so  good. 


Original  Method  of  Artificial  Parthej 


ENOGENESIS      169 


which  crops  up  again  and  again  in  provoking  the  development 
of  eggs  by  hypertonic  solutions:  it  is  that  the  eggs  develop  only 
if  the  supply  of  oxygen  in  the  hypertonic  solution  is  sufficient. 
If  they  lie  thickly  upon  one  another,  a  mutual  struggle  for 
oxygen  takes  place,  and  the  hypertonic  solution  remains  with- 
out effect.  The  same  happens  when  the  eggs  are  covered  with 
too  deep  a  la3Tr  of  water,  which  prevents  a  rapid  diffusion  of 
oxygen  into  them. 

5.  If  unfertilized  eggs  remain  longer  in  the  hypertonic  sea- 
water  than  is  necessary  for  their  development,  they  will  disin- 
tegrate when  put  back  into  normal  sea-water.     After  transfer- 
ence into  normal  sea-water  they  break  up  into  small  droplets; 
they  do  not  undergo  such  a  change  while  they  are  in  the  hyper- 
tonic solution.     This  destructive  effect  of  the  hypertonic  solu- 
tion can  be  inhibited  by  depriving  the  hypertonic  solution  of 
oxygen  or  by  adding  a  trace  of  KCN  or  of  chloral  hydrate  to 
the  sea-water.  1     Fertilized  eggs  suffer  more  rapidly  in  the  hy- 
pertonic solution  than  unfertilized  eggs,  and  they  can  also  be 
protected  against  the  toxic  action  of  the  hypertonic  solution  by 
the  suppression  of  the  oxidations  in  the  egg.     The  reason  that 
fertilized  eggs  are  injured  more  rapidly  by  the  hypertonic  solu- 
tion than  the  unfertilized  eggs  is  probably  due  to  the  fact 
that  the  rate  of  oxidations  is  so  much  greater  in  the  fertilized 
than  in  the  unfertilized  eggs.     These  facts  have  already  been 
discussed  in  a  previous  chapter. 

Warburg  assumes  that  the  oxidations  are  accelerated  ex- 
cessively by  the  hypertonic  solution.  But  this  is  not  the  case  for 
the  fertilized  eggs  of  S.  pnrpuratus  on  which  these  experiments 
were  carried  out,  since  Wasteneys  and  the  writer  found  that  the 
rate  of  oxidations  in  the  fertilized  eggs  of  S.  pnrpuratus  is  not 
accelerated  by  the  hypertonic  solution.  Either  the  oxidations 
are  modified  by  the  hypertonic  solution  so  as  to  lead  to  the 

iLoeb,  "Ueber  die  Hemmung  der  toxischcn  Wirkung  dcr  In  pertonischcn 
Losungen  auf  das  Seeigelei  durch  Saucrstoffniangol  und  Cyankalium."  Pfluaer't 
Archiv,   CXIII,  487,    1906. 


170    Artificial  Parthenogenesis  and  Fertilization 


formation  of  abnormal  chemical  products  in  the  egg;  or  the 
oxidations  lead  to  phj'sical  or  morphological  modifications  in 
the  egg,  which  cause  its  disintegration  when  it  is  put  back 
into  normal  sea-water.  We  can  imagine  that  chloral  hydrate 
acts  favorably  by  suppressing  certain  morphological  changes 
in  the  egg. 

While  it  is  not  always  possible  to  induce  artificial  partheno- 
genesis by  the  purely  osmotic  method,  the  destruction  of  the 
eggs  by  this  method  and  the  prevention  or  retardation  of  this 
destruction  by  lack  of  ox^^gen  always  succeeds. 

6.  In  1908  Delage  published  a  method  of  artificial  partheno- 
genesis which  gave  good  results.  The  unfertilized  eggs  were 
put  into  a  mixture  of  50  c.c.  of  a  cane-sugar  solution  and  sea- 
water  to  which  he  added  23  drops  of  a  N/10  tannic-acid  solution 
and  30  drops  of  a  N/10  solution  of  NH4OH.  The  concentra- 
tion of  the  sugar  solution  was  1 .  135  N.  He  used  15  c.c.  sea- 
water  and  35  c.c.  cane-sugar  solution.^  In  reality  this  method 
is  identical  with  my  method  of  combining  alkaline  and  hy- 
pertonic solutions  simultaneously.  The  1 .  135  N  sugar  solution 
is  strongly  hypertonic  (see  chap.  xiii).  The  ammonia  is  in 
excess  of  the  tannic  acid  and  NH4OH  is,  as  we  have  seen,  one 
of  the  bases  which  diffuses  easily  into  the  egg,  and  hence  is 
very  efficient  in  the  production  of  artificial  parthenogenesis. 

Since  any  hypertonic  solution  with  the  proper  amount  of 
NH4OH  acts  in  the  same  way  as  Delage's  solution,  neither 
the  presence  of  tannic  acid  nor  of  sugar  is  essential. 

Shearer  and  Miss  Lloyd^  have  used  Delage's  mixture  in  the 
place  of  the  hypertonic  sea-water  in  my  ''improved"  method. 
They  produced  membrane  formation  in  the  unfertilized  eggs 
of  Echinus  with  butyric  acid.  Instead  of  putting  them  into 
hypertonic  sea-water  afterward,  they  put  them  for  one  hour 

1  Delage,  "Les  vrais  facteurs  de  la  parthenogenese  experimentale,"  Arch,  de 
Zool.  exper.  et  gen.,  4me  ser.,  VII,  446,  1908. 

2  Shearer  and  Miss  Lloyd,  Quarterly  Jour.  Microscopical  Science,  LVIII,  523, 
1913. 


Original  Method  of  Artificial  Parthenogenesis     171 


into  Delage's  mixture  which  is  merely  a  hypertonic  solution 
rendered  alkaline  by  an  excess  of  NH4OH.  It  is  obvious  that 
in  this  case  the  solution  of  Delage  could  have  produced  only 
the  corrective  effect  typical  for  hypertonic  solutions.  The  addi- 
tional NH4OH  could  do  no  good,  and  probably  did  some  harm. 
The  larvae  which  were  produced  by  this  method  did  not  de- 
velop as  far  as  those  produced  when  the  simple  hypertonic  sea- 
water,  as  I  use  it,  was  added. 

Delage  has  drawn  the  conclusion  that  in  his  experiments 
the  tannic  acid  causes  a  coagulation  and  the  alkali  a  liquefac- 
tion, and  that  development  is  due  to  an  alteration  of  coagula- 
tions and  hquefactions.  Since  we  have  shown  in  this  and  the 
previous  chapter  that  the  mere  treatment  of  the  eggs  with  an 
alkaline  hypertonic  solution  causes  the  production  of  parthe- 
nogenetic  plutei,  and  since  it  can  be  demonstrated  that  any 
hypertonic  solution  containing  the  proper  amount  of  NH4OH 
or  any  other  weak  base  acts  as  well  as  Delage's  solution,  the 
conclusion  he  draws  from  his  observation  becomes  untenable. 


XVII 
MEMBRANE  FORMATION  AND  CYTOLYSIS 

1.  In  this  chapter  we  shall  show  that  all  haemolytic  agents 
also  cause  membrane  formation.  We  find  usually  that  a  short 
exposure  of  the  egg  to  such  a  reagent  leads  only  to  membrane 
formation,  a  longer  one  to  cytolysis.  According  to  Koppe, 
besides  electricity  and  heat,  there  are  five  classes  of  reagents 
that  cause  cytolysis;  they  are:  (1)  certain  specific  substances, 
such  as  glucosides  (e.g.,  saponin)  or  bile  salts;  (2)  a  number 
of  fat-solvents,  such  as  benzol,  ether,  and  alcohol;  (3)  distilled 
water;  (4)  acids;  (5)  bases.  We  have  already  seen  that  the 
two  last-named  substances  lead  to  membrane  formation,  and 
we  shall  show  that  the  same  is  true  for  the  others,  and  that  with 
proper  after-treatment  this  membrane  formation  leads  to  the 
development  in  the  sea-urchin  egg  (and  the  eggs  of  other 
forms) .  We  can  therefore  say  briefly  that  all  haemolytic  agencies 
effect  the  activation  of  the  unfertilized  egg,  and  this  activation  con- 
sists in  a  cytolysis  of  the  cortical  layer  of  the  eggs. 

I  first  noticed  the  connection  between  membrane  formation 
and  cytolysis  when  in  1904  I  was  trying  to  discover  why  no 
typical  membrane  was  formed  in  my  original  method  of  artificial 
parthenogenesis  by  hypertonic  solutions.  In  the  course  of  these 
investigations  it  happened  that  when  the  osmotic  pressure  was 
high  enough,  e.g.,  with  1 J  m  solutions  of  XaCl  or  cane  sugar,  the 
unfertilized  eggs  produced  a  splendid  fertilization  membrane; 
but  this  membrane  formation  was  followed  almost  at  once  by 
a  cytolysis  of  the  whole  egg.^  A  similar  phenomenon  occurs 
when  the  eggs  are  placed  in  distilled  water;  for  they  also  form 
membranes  and  are  immediately  afterward  transformed  into 
"shadows." 

1  Loeb,   "Ueber  Befruchtung,  kiinslliche  Parthenogenese  and  Zytolyse  cles 
Seeigeleies,"  Pfliluers  Archiv,  XCIII,  257.  1904;     Utitersuchunaen,  p.  28s/ 

173 


174     Artificial  Parthenogenesis  and  Fertilization 

Shortly  afterward  my  attention  was  again  drawn  to  the 
connection  between  membrane  formation  and  cytolysis,  when 
performing  experiments  upon  the  effect  of  benzol  and  amylene 
upon  the  unfertilized  sea-urchin  eggs.  Here  too  it  turned  out 
that  the  first  effect  of  these  hydrocarbons  was,  as  Herbst^  had 
already  observed,  the  formation  of  a  fertihzation  membrane, 
but  this  was  followed  almost  at  once  by  the  cytolysis  of  the  egg.^ 
I  found  that  if  the  eggs  are  removed  soon  enough  from  the 
sea-water  containing  benzol  or  amylene,  they  can  be  caused  to 
develop  into  normal  larvae  after  a  short  treatment  with  a  hyper- 
tonic solution.  A  similar  result  w^as  obtained  in  experiments 
upon  the  artificial  meml^rane  formation  by  the  higher  fatty 
acids,  and  we  shall  return  to  this  again.' 

We  will  now  discuss  the  series  of  different  cytolytic  reagents 
from  the  point  of  view  of  their  membrane-forming  effect,  and 
we  will  start  with  the  first  group,  that  of  the  specific  haemolytic 
substances  such  as  the  glucosides  (saponin,  solanin,  digitalin), 
bile  salts,  and  soaps;  in  this  group  belong  the  foreign  sera, 
to  the  consideration  of  which  a  special  chapter  will  be  devoted. 
We  shall  start  with  a  description  of  the  effect  of  saponin  upon 
the  sea-urchin  egg. 

2.  Figs.  39  to  45  are  camera  drawings  of  the  changes  of  an 
egg  in  a  saponin  solution  (8  drops  of  a  i  of  1  per  cent  saponm 
solution  in  sea-water  to  5  c.c.  of  sea-water).  Fig.  39  gives  the 
size  and  the  appearance  of  the  egg  immedia,tely  after  trans- 
ference to  the  saponin  solution  (at  9:07  a.m.).  Membrane 
formation  (Figs.  40  and  41)  started  four  minutes  afterward, 
and  at  Fig.  42  a  normal  fertilization  membrane  had  been 
formed.  Five  minutes  later,  cytolysis  starts,  and  indeed  in 
this  case  it  depends  upon  a  process  which  superficially  and 
in  the  beginning  resembles  membrane  formation.     For  at  G 

1  Herbst,   Biol.   Centralbl.,  XIII,   14,   1893. 

2  Loeb,  "Ueber  eine  verbesserte  Methode  der  kiinstlichen  Parthenogenese," 
Untersuchungen,  p.  340. 

3  Ibid.,  p.  342. 


Membrane  Formation  and  Cytolysis 


175 


Fig.  39 


Fig.  40 


Fig.  41 


Fig.  42 


Fig.  43 


Fig.  44 


Fig.  45 


Formation  of  the  fertilization  membrane  and  cytolysis  of  the  sea-urchin  egg  on 

treatment  with  saponm. 

YiG.   39.— Appearance  of  egg  when  first  exposed  to  saponin  solution  at 

^■^Vi*G^'  40  41,  and  42.— Formation  of  fertilization  membrane.  The  stage  of 
comoleted  membrane  formation  as  depicted  in  Fig.  42  was  reached  at  9:lo  a.m. 
af  at  this  stage  the  egg  is  withdrawn  from  the  influence  of  the  saponin  it  can 
rit^veloD  )     Cytolysis  (Fig.  43)  began  at  9:20  a.m.         .    .    ^        .      .^, 

Figs.  43.  44.  and  45.— Advanced  stages  of  cytolysis  .nduced  with  saponin. 


176     Artificial  Parthenogenesis  and  Fertilization 

in  Fig.  43  there  occurs  an  outflow  of  clear  matter  from  the 
cytoplasm,  just  as  in  membrane  formation.  This  is  quickly 
followed  by  the  clearing  and  swelling  of  the  whole  egg,  until  at 
last  it  becomes  a  '^ shadow"  (Figs.  44  and  45).  The  variations 
that  present  themselves  on  examination  of  a  quantity  of  eggs 
in  the  saponin  solution  occur  principally  in  the  membrane 
formation.  In  many  cases  the  formation  of  vesicles,  which  is 
here  depicted  in  Figs.  40  and  41,  did  not  take  place.  Instead 
of  this,  the  egg  proceeds  from  the  stage  drawn  in  Fig.  39  directly 
into  the  stage  of  membrane  formation  (Fig.  42).  As  an  inter- 
mediate stage  one  observes  cases  in  which  onl}'  a  roughening 
of  the  surface  of  the  egg  takes  place,  owing  to  the  formation 
of  numerous  minute  drops,  which  suddenly  swell  simultaneously 
and  give  rise  to  the  formation  of  the  surface  membrane;  at  first 
this  is  closely  adjacent  to  the  cytoplasm,  but  as  a  result  of  the 
osmotic  pressure  more  sea-water  is  always  flowing  in,  until  the 
tension  of  the  membrane  counterbalances  the  osmotic  pressure. 

The  examination  of  these  drawings  also  gives  the  impres- 
sion that  the  effect  of  the  saponin  is  produced  in  two  distinct 
stages,  w^iich  do  not  necessarily  merge  continuousl}^  into  each 
other;  the  first  stage  is  probably  the  effect  upon  the  surface  of 
the  egg,  leading  to  membrane  formation,  and  the  second  is 
obviously  an  effect  produced  upon  the  interior  of  the  egg,  and 
leads  to  cytolysis.  In  both  cases  the  liquefaction  is  associated 
with  a  swelling  and  increase  in  volume. 

When  eggs  that  have  been  treated  with  saponin  until 
membrane  formation  has  taken  place  are  freed  from  the  last 
trace  of  saponin  by  washing  them  four  to  six  times  in  pure 
sea-water,  they  behave  just  like  eggs  in  which  the  artificial 
membrane  has  been  produced  by  a  fatty  acid.  If  the}'  are  left 
in  sea-water,  development  starts,  and  the  stage  to  which  they 
develop  depends  on  the  temperature.  If  they  are  subsequently 
treated  for  about  forty  minutes  (at  15°  C.)  with  hypertonic 
sea-water,  they  develop  into  larvae. 


Membrane  Formation  and  Cytolysis  177 


The  following  example  will  illustrate  this.  Four  drops  of  a 
weak  solution  of  saponin  in  sea-water  were  added  to  5  c.c.  of 
sea-water  with  eggs.  Membrane  formation  started  after  five 
minutes,  and  three  minutes  later  all  the  eggs  possessed  mem- 
branes. The  eggs  were  then  washed  in  200  c.c.  of  sea-water  that 
was  free  from  saponin,  and  this  washing  was  repeated  four  times. 
They  were  next  put  into  50  c.c.  sea-water +6.5  c.c.  of  2§  m  XaCl. 
Portions  of  the  eggs  were  replaced  in  sea-water  after  15,  25,  33, 
45,  55,  65,  and  93  minutes,  respectively.  All  eggs  that  had  been 
only  15,  25,  or  35  minutes  in  hypertonic  sea-water  disintegrated. 
A  few  of  the  eggs  that  had  been  45  minutes  in  the  hypertonic 
solution  developed  into  larvae,  while  10  per  cent,  60  per  cent, 
and  80  per  cent  of  larvae  were  given  by  the  eggs  that  had  been 
55,  65,  and  93  minutes,  respectively,  in  the  hypertonic  solution. 

As  a  control  I  treated  eggs  in  the  same  manner  after  mem- 
brane formation  by  means  of  butyric  acid;  similar  results  were 
obtained.  The  life  duration  of  the  butyric-acid  larvae  was, 
however,  longer  than  that  of  the  saponin  larvae.^ 

We  know  that  solanin  and  digitalin  possess  haemolytic 
properties  like  saponin.  It  can  also  be  shown  that  sea-urchin 
eggs  that  have  been  induced  to  form  membranes  by  these 
agencies  can  be  caused  to  develop  into  larvae. 

The  cytolytic  effect  of  bile  salts  is  well  known.  A  mixture 
of  sodium  glycocholate  and  taurocholate  was  dissolved  in  water. 
Membrane  formation  soon  followed  when  eggs  were  placed  in 
such  a  solution.  The  membrane  was  formed  while  the  eggs 
were  still  in  the  solution  of  the  bile  salts,  just  as  in  the  case  of 
the  saponin  solution.  Membrane  formation  was  rapidly  fol- 
lowed by  cytolysis  of  the  egg.  But  if  the  eggs  were  transferred 
to  normal  sea-water  in  time,  cytolysis  did  not  supervene.  On 
being  transferred  at  th(^  right  time  (i.e.,  after  a  membrane  had 
formed,  but  before  they  had  all  cytolyzed)  from  the  solution  of 

1  Loeb,  "Ueber  die  Hervorrufung  dor  MembranbildunK  mid  Eiitwickhing 
beim  Seeigolei  durch  Blutseniiu  von  Kaninclua  uud  durch  zytolytischo  Agenzien." 
Pfluger's  ^Archiv,   CXXII,    199,    1908. 


178    Artificial  Parthenogenesis  and  Fertilization 

bile  salts  to  normal  sea-water,  they  did  not  develop  if  left 
therein.  But  some  of  them  did  develop  into  larvae  after  a 
subsequent  short  exposure  to  a  hypertonic  solution.  The 
eggs  were  more  harmed  by  membrane  formation  with  bile  salts 
than  by  membrane  formation  with  saponin. 

In  both  cases  only  those  eggs  developed,  after  a  short  treat- 
ment with  hypertonic  sea-water,  which  had  formed  membranes, 
a  fact  which  is  also  true  of  eggs  treated  with  a  fatty  acid.^ 

3.  In  1905  I  started  experiments  on  membrane  formation  in 
the  egg  by  means  of  soaps;  but  by  chance  these  experiments 
led  to  no  results.  The  experiments  were  resumed  a  few  years 
later  with  positive  results.  For  since  soaps  are  good  cytolytic 
agents,  one  could  postulate  a  definite  result  from  these  experi- 
ments. I  will  first  briefly  portray  the  c>i:olytic  effect  of  the 
soaps  and  then  discuss  their  developmental  effect. 

As  soap  is  precipitated  by  calcium,  it  was  necessary  to 
dissolve  it  in  m/2  NaCl  solution,  instead  of  in  sea-water.  Now 
when  the  unfertilized  eggs  of  S.  purpuratus  (after  being  freed 
from  sea-water  by  washing  in  m/2  NaCl)  are  placed  in  50  c.c. 
of  neutral  m/2  NaCl +2  c.c.  m/10  sodium  oleate,  neither  mem- 
brane formation  nor  cytolysis.  occurs  as. a  rule  in  that  solution. 
The  eggs  only  become  angular.  But  if  they  are  placed  after  a 
few  minutes  in  sea-water,  a  large  number  of  them  form  mem- 
branes at  once,  which  in  a  few  eggs  is  followed  by  cytolysis. 
The  longer  the  eggs  remain  in  the  soap  solution,  the  greater 
becomes  the  percentage  forming  membranes  and  cytolyzing 
after  transference  to  ordinary  sea-water.  For  the  eggs  of  many 
sea-urchins  the  addition  of  2  c.c.  of  sodium  oleate  to  50  c.c.  of 
NaCl  is  too  little,  and  about  3  c.c.  of  soap  must  be  added. 

Why  does  the  soap  solution  not  cause  membrane  formation 
until  after  the  egg  has  been  replaced  in  sea-water  ?  The  reason 
is  that  it  is  the  sea-water  which  causes  the  membrane  forma- 
tion as  a  result  of  its  alkaline  reaction.     The  soap  solution  either 

1  Loeb,  op.  cit. 


Membrane  Formation  and  Cytolysis  179 


makes  the  egg  more  permeable  to  alkali  or  the  effect  of  the  two 
substances  is  cumulative.     This  can  be  demonstrated  by  a  very 
striking  series  of  experiments.     Unfertilized  eggs  were  placed 
in  50  c.c.  of  m/2  NaCl+2  c.c.  of  m/10  sodium  oleate.     Three 
and  one-half  minutes  later  some  of  them  were  replaced   in 
sea-water.     The  majority  of  these  eggs  formed  membranes  at 
once,    and    subsequently   many   succumbed    to    cytolysis.     A 
second  portion  of  the  eggs  was  transferred  to  50  c.c.  of  sea- 
water  slightly  acidified  by  the  addition  of  0.5  c.c.  N/10  HCl 
(0.4  c.c.  HCl  is  enough  to  produce  a  change  in  color  of  neutral 
red  in  sea-water).     Not  an  egg  that  had  been  transferred  to 
this  shghtly  acid  sea-water  formed  a  membrane  or  cj-tolyzed. 
The  addition  of  even  0.3  c.c.  or  still  less  HCl  was  enough  to 
prevent  membrane  formation  and  cytolysis  of  the  eggs  after 
they  had  been  transferred  to  sea-water.     On  the  other  hand, 
the  cytolytic  effect  of  the  sea-water  was  greatly  increased  by 
the  addition  of  alkali.     The  following  experiment  may  serve 
as  an  example.     Unfertilized  eggs  were  placed  in  50  c.c.  m/2 
NaCl+0.2  c.c.  m/10  sodium  oleate  solution.     Three  minutes 
later,  they  were  transferred  to  the  following  solutions:   50  c.c. 
of  sea-water+0.5,  0.3,  0.1,  0  c.c.  N/10  HCl;  0.5,  1.0,  1.5 
c.c.    N/10  NaOH.     Among   the   eggs   that   had   been   trans- 
ferred to  the  sea-water  to  which  acid  had  been  added  none 
or  extremely   few   formed   membranes,  and  no  cytolysis  oc- 
curred.     Of  the   eggs   transferred   to   ordinary   sea-water  95 
per  cent  formed  membranes,  and  about  1  per  cent  of  them 
cytolyzed.     All  the  eggs  that   were   transferred  to  the  sea- 
water+0.5  c.c.  N/10  NaOH  formed  membranes,  and  90  per 
cent  of  them    cytolyzed.      In  the  cases   where   more   NaOH 
had  been  added  to  the  sea-water  all  the  eggs  fell  victims  to 
cytolysis. 

If  our  line  of  thought  is  correct,  it  must  also  be  possible  to 
cause  membrane  formation  and  cytolysis  in  the  eggs  while  they 
are  still  in  the  soap  solution,  by  merely  making  it  alkaline.     And 


180     Artificial  Parthenogenesis  and  Fertilization 


this  is  actual!}'  the  case.    Thus  unfertiHzed  eggs  were  distributed 
among  the  following  solutions: 

(1)  25  c.c.  m/2  NaCl-hO.  1  c.c.  m/10  sodium  oleate 

(2)  25  c.c.  m/2  NaCl+0. 1  c.c.  m/10  sodium  oleate+0.2  c.c.  N/10 

NaOH 

(3)  25  c.c.  m/2  NaCl+0.2  c.c.  N/10  NaOH. 

In  the  first  solution,  the  eggs  became  angular,  but  hardly 
one  formed  a  membrane;  in  the  second  solution,  50  per  cent  of 
the  eggs  formed  very  delicate  membranes  that  soon  tore,  and 
many  of  the  eggs  cytolyzed;  in  the  third  case  the  eggs  remained 
quite  intact. 

But  cytolysis  of  the  eggs  in  the  soap  solution  can  also  be 
obtained  by  raising  the  concentration  of  the  soap  solution  high 
enough.  Thus,  if  the  eggs  are  put  into  50  c.c.  m/2  NaCl+1  c.c. 
m/10  sodium  oleate,  membrane  formation  and  cytolysis  begin 
practically  at  once.  I  suppose  such  a  solution  has  a  faintly 
alkaline  reaction.  If  excess  of  HCl  is  added  to  this  mixture, 
membrane  formation  is  not  prevented;  in  this  case  free  oleic 
acid  is  formed,  and  this,  as  I  observed  in  1905,  also  initiates 
membrane  formation  and  cytolysis  in  the  egg. 

We  have  already  seen  that  an  alkaline  solution  of  NaCl 
without  soap  has  only  a  relatively  weak  cytolytic  effect.  The 
addition  of  0.2  c.c.  of  m/10  sodium  oleate +0.4  c.c.  N/10 
NaOH  to  50  c.c.  of  NaCl  has  just  as  much  effect  as  the  addi- 
tion of  2  c.c.  NaOH  without  soap. 

Now  it  is  easy  to  convince  oneself  of  the  effect  of  soap  in 
causing  development.  It  is  only  necessary  to  transfer  the  eggs 
after  a  short  time  from  the  soap  solution  to  ordinary  sea-water, 
and  subsequently  (after  repeated  washing)  expose  them  to 
hypertonic  sea-water.  The  following  example  will  illustrate 
this:  Unfertilized  eggs  were  placed  in  50  c.c.  m/2  NaCl  + 
0.2  c.c.  of  sodium  oleate  and  after  two  or  three  minutes  trans- 
ferred to  sea-water.  The  majority  of  the  eggs  formed  mem- 
branes and  only  a  few  cytolyzed.      The  eggs  were  washed 


Membrane  Formation  and  Cytolysis  181 

repeatedly,  and  one  hour  later  placed  in  hj'pertonic  sea-water 
from  which  they  were  transferred  to  ordinary  sea-water  after 
thirty  to  fifty  minutes.  A  number  of  the  eggs  which  had 
formed  membranes  developed  into  larvae. 

These  experiments  prove  that  the  membrane  formation 
induced  by  soaps  can  also  set  up  development.  This  method, 
however,  has  little  to  recommend  it  for  practical  purposes, 
because  the  cytolytic  effect  of  the  soap  is  so  strong.  Eggs 
treated  with  soap  show  a  much  greater  tendency  to  cytolysis 
than  those  treated  with  a  low  fatty  acid. 

4.  Another  group  of  haemolytic  agents  is  formed  by  fat- 
solvents,  such  as  benzol,  toluol,  amylene,  chloroform,  aldehyde, 
ether,  alcohol,  etc.  It  has  already  been  mentioned  that  when 
benzol,  toluol,  and  amylene  are  dissolved  in  sea-water — only 
a  trace  of  them  is  soluble — they  produce  a  membrane  forma- 
tion in  eggs,  which  is  followed  practically  at  once  by  cytolysis. 
Hence  the  specific  fat-solvents  are  of  little  use  for  artificial 
parthenogenesis.  The  same  principle,  however,  that  is  found 
in  the  saponin  group  also  applies  here:  by  working  quickly, 
removing  the  eggs  from  the  benzol  or  amylene  sea-water  as  soon 
as  membrane  formation  has  taken  place,  and  transferring  them 
to  ordinary  sea-water,  it  can  be  demonstrated  that  a  certain 
percentage  of  eggs  form  membranes,  without  undergoing 
cytolysis.  These  eggs  can  be  caused  to  develop  into  larvae 
by  treating  them  subsequently  with  hypertonic  sea-water.  If 
they  are  not  treated  with  hypertonic  sea-water,  they  do  l)egin 
to  develop,  but  they  disintegrate  before  reaching  the  larval 

stage. 

Owing  to  the  importance  of  this  subject,  w^e  will  describe  the 
cytolysis  of  the  sea-urchin  egg  under  the  influence  of  a  reagent 
belonging  to  this  group.  Figs.  46  to  51  show  the  behavior  of 
the  sea-urchin  egg  in  a  mixture  of  45  c.c.  of  sea-water -|-5  c.c. 
of  m/100  salicyl  aldehyde.  A  beautiful  membrane  formation 
first  takes  place  (Fig.  49);    but  then  cytolysis  starts  with  the 


l!?_^5^^frciAL  Parthenogex 


Esis  AND  Fertilization 


i^'ig.  46 


Fig.  48 


Fig.  47 


Fig.  49 


Fig.  50 
-Membrane  formation 


Fig.  51 


uence  of 


'SIS. 


Membrane  Formation  and  Cytolysis 


183 


outflow  of  a  clear  substance  from  the  cytoplasm  (Fig.  50), 
and  soon  after  the  egg  is  changed  to  a  shadow  (Fig.  51). 

In  this  case  also  the  process  of  cytolysis  is  divided  into  two 
stages,  at  first,  a  membrane  formation,  and  soon  after  a  complete 
clarification  of  the  interior  and  the  formation  of  large  clear 
drops  (Fig.  51). 

If  the  eggs  are  placed  in  a  mixture  of  10  c.c.  of  sea-water + 
10  c.c.  2i  m  propylalcohol,  the  formation  of  a  fertihzation  mem- 
brane is  the  first  thing  that  takes  place.     If  the  eggs  are  removed 


Fig.  52  Fig.  5;i 

Figs.  52  and  53. — Membrane  formation  and  cytolysis  in  two  different  eggs 
of  a  sea-urchin  under  the  influence  of  propylalcohol.  In  Fig.  52  cytolysis  starts 
with  the  outflow  of  many  small  droplets;  in  Fig.  53  with  the  outflow  of  only  one 
large  drop  from  the  cytoplasm. 

from  the  solution  at  this  stage,  and  washed  with  sea-water,  a 
few  are  able  to  develop  after  a  subsequent  exposure  to  hyper- 
tonic sea-water. 

But  if  the  eggs  are  left  in  the  solution  of  alcohol,  cytolysis 
quickly  ensues.  Figs.  52  and  53  show  the  start  of  this  cytolysis 
which  in  the  one  case  consists  in  the  outflow  of  many  clear  drops 
from  the  egg,  and  in  the  other  of  one  large  drop.  At  bottom, 
it  only  depends  upon  two  modifications  of  the  same  process. 
It  may  be  mentioned  that  the  result  of  the  experiment  remains 
the  same  if  1  c.c.  of  2J  m  NaCl  is  added  to  each  4  c.c.  of  propyl- 
alcohol. 

5.  We  come  now  to  a  discussion  of  the  cytolytic  action  of 
distilled  water.     This  type  of  cytolysis  is  of  great  impoitance 


184     Artificial  Parthenogenesis  and  Fertilization 


from  a  theoretical  point  of  view.  The  drawings  in  Figs,  54 
to  58  depict  the  behavior  of  the  unfertiHzed  sea-urchin  egg  in 
distilled  water.  It  will  be  seen  that  during  the  first  five  minutes 
the  egg  slowly  but  steadily   increases  in  volume  though   its 


Fig.  54 


Fi^.  55 


Fig.  56 


Fig.  57 


Fig.  58 


Figs.  54-58. — Swelling  and  cytolysis  of  the  sea-urchin  egg  in  distilled 
water.  Slow  but  steady  increase  in  volume  of  the  egg  in  distilled  water  during 
a  period  of  five  minutes  (Figs.  54-57).  Instantaneous  membrane  formation, 
swelling  and  cytolysis  of  the  egg  within  the  sixth  minute  (Fig.  58).  It  is  obvious 
that  the  cytolysis  is  not  caused  by  a  biu-sting  of  the  surface-layer  of  the  egg. 

appearance  remains  the  same  (Figs.  55  to  57).  Then  it  is 
quite  suddenly  converted  into  a  shadow  within  one  minute 
(Fig.  58);  in  that  time  it  forms  a  membrane  and  swells  enor- 
mously. Hence  a  change  of  condition  must  occur  in  that 
minute.  At  first  the  egg  is  in  possession  of  a  semi-permeable 
membrane  which  is  only  permeable  for  water,  but  not  for  salts. 
In  this  condition  the  changes  noted  in  Figs.  54  to  57  occur. 


Membrane  Formation  and  Cytolysis  185 

Then  the  egg  becomes  permeable  for  salts  but  not  for  colloids. 
Then  the  sudden  swelling  in  Fig.  58  takes  place.  We  shall 
return  to  this  phenomenon  in  chap.  xx. 

6.  Increase  of  temperature  also  produces  cytolysis.  Dr. 
von  Knaffl  found  that  heating  unfertilized  sea-urchin  eggs  to 
41°  C.  led  to  their  practically  instantaneous  cytolysis.  At 
lower  temperatures  a  longer  time  is  necessary  for  cytolysis.^ 
I  found  that  by  merely  warming  sea-urchin  eggs  to  34°  or  35°  C, 
the  formation  of  a  typical  fertilization  membrane  can  often, 
but  not  always,  be  induced.^  If  the  eggs  are  then  cooled 
quickly,  no  cytolysis  follows.  Such  eggs  are  no  longer  capable 
of  development,  since  a  temperature  of  34°  C.  kills  them. 
But  starfish  eggs,  which  can  also  be  caused  to  form  membranes 
by  warming,  can  endure  a  higher  temperature,  and  develop 
under  these  conditions  (according  to  experiments  of  Ralph  S. 
Lillie,  which  we  shall  discuss  later). 

7.  We  will  now  return  to  a  short  discussion  of  the  effect 
of  acids.  We  have  already  described  in  detail  how  the  acids 
cause  membrane  formation.  As  far  as  cytolysis  of  the  sea- 
urchin  egg  is  concerned,  I  have  obtained  it  only  with  the  higher 
fatty  acids,  beginning  with  heptylic,  i.e.,  with  heptylic,  caprylic, 
nonylic,  and  capric  acids;  this  cytolysis  is  always  preceded  b>' 
the  formation  of  a  fertilization  membrane.  The  cytolysis 
takes  place  while  the  eggs  are  in  the  solution.  The  experi- 
ments were  usually  performed  in  N/500  or  N/ 1,000  solution  of 
the  acid,  which  was  rendered  isosmotic  with  sea-water  by  the 
addition  of  some  NaCl.  Curiously  enough,  the  addition  of 
some  Ca  promoted  the  membrane  formation  in  a  very  marked 
fashion.  Oleic  acid  (rendered  isotonic  with  sea-water  by  NaCl) 
also  caused  membrane  formation  and  c^'-tolysis  in  the  sea-urchin 
egg.  With  the  lower  fatty  acids,  from  capronic  downward,  1 
have   so    far    obtained    membrane    formation,    but    no    rapid 

1  Von  Knaffl,   Pfluger's   Archiv,  CXXIII,  279.   1908. 

2  Loeb,  Pfliiger's  Archiv,  CXXII,  199.  1908. 


186    Artificial  Parthenogenesis  and  Fertilization 

cytolysis.  Can  it  be  possible  that  the  lower  fatty  acids  are 
only  soluble  in  the  cortical  Ia\er  of  the  unfertilized  egg  while 
the  higher  fatty  acids  are  soluble  in  the  whole  egg  and  hence 
cause  cytolysis  ? 

8.  During  experiments  (which  will  be  discussed  later  in 
this  book)  on  the  sensitizing  effect  of  SrCl2  and  BaCU  upon  the 
eggs  of  the  sea-urchin,  the  writer  found  that  unfertilized  eggs 
of  S.  purpuratus  and  franciscanus  when  left  in  isotonic  (3/8  m) 
solutions  of  SvCU  or  BaCl2  would  sooner  or  later  form  typical 
fertilization  membranes.  The  time  required  for  this  effect 
varied  considerably  for  the  eggs  of  various  females.  In  some 
cases  eggs  would  form  membranes  in  ten  minutes,  in  other 
cases  just  as  manj^  hours  were  required.  When  such  eggs 
were  left  to  themselves,  they  disintegrated  like  the  eggs  in 
which  membrane  formation  was  produced  by  butyric  acid. 
When  afterward  treated  with  a  hypertonic  solution  for  a  short 
time  the  eggs  would  develop  into  larvae.^ 

The  writer  tried  vainly  to  produce  similar  effects  by  the 
pure  salts  of  NaCl,  Na2S04  or  sodium  oxalate,  and  many  other 
salts.  The  unfertilized  eggs  of  *S.  purpuratus  will  live  in  an 
m/2  NaCl  solution  for  several  daj^s  without  suffering  any  injury 
and  the  same  was  found  true  for  solutions  of  CaCla.  When 
treated  with  sperm  the  eggs  could  be  fertilized  and  they  would 
develop  normally. 

This  resistance  of  the  eggs  of  purpuratus  to  isotonic  sodium 
salts  is,  however,  quite  extraordinary.  It  does  not  exist  in  the 
egg  of  Arbacia.  In  these  eggs,  R.  Liliie^  succeeded  in  calling 
forth  membrane  formation  by  putting  them  for  five  or  ten 
minutes  into  pure  solutions  of  Nal  or  KI  or  NaCNS  and  KCNS, 
and  then  transferring  them  to  sea- water.  ''A  large  proportion, 
in  favorable  experiments  practically  all,  form  fertilization  mem- 
branes, usually  thin  and  close  to  the  egg  surface."     These  eggs 

1  Loeb,   Archiv  f.   E ntwicklungsmechanik,  XXX,  44,   1910. 

2R.  Lillie,  "The  Physiology  of  Cell  Division,"  Jour.  Morphol.,  XXII,  695, 
1911. 


Membrane  Formation  and  Cytolysis  187 


begin  to  segment,  but  sooner  or  later  disintegrate  though  a 
few  may  reach  an  early  blastula  stage.  Such  eggs  are,  however, 
abnormal.  But  if  these  eggs  are  exposed  for  a  short  time  (in 
Lillie's  experiments  thirty  minutes)  to  a  hypertonic  solution  a 
large  number  will  develop.  The  membrane  formation  produced 
by  these  salts  had  therefore  the  same  effect  as  treatment  of  the 
eggs  with  fatty  acids. 

Lillie  made  the  very  interesting  observation  that  the 
addition  of  some  CaCls  to  the  solution  of  Nal  inhibited  the 
membrane  formation.  The  following  experiments  illustrate  this 
result.  Unfertilized  eggs  of  Arhacia  were  put  for  five  minutes 
into  250  c.c.  0.55  m  Nal;  when  transferred  to  sea-water  all 
formed  membranes.  Eggs  left  for  five  minutes  in  250  c.c. 
0.55  m  Nal +15  c.c.  m/2  CaClg  practically  all  remained  normal 
when  put  back  into  sea-water.  The  solution  containing  Ca  is, 
however,  not  entirely  ineffective,  since  some  eggs  treated  with 
this  solution  may  develop  into  larvae  when  afterward  exposed 
for  a  short  time  to  a  hypertonic  solution. 

R.  LilHe  explains  his  results  on  the  assumption  that  the 
effective  salts  increase  the  permeability  of  the  egg,  and  that 
this  rise  in  permeabihty  is  checked  through  the  addition  of 
CaCla-  The  primary  effect  is  probably  a  cytolytic  action  upon 
the  cortical  layer  of  the  egg,  and  on  this  assumption  the  observa- 
tions of  Lillie  on  the  membrane  formation  by  NaT  and  NaCNS 
harmonize  with  the  observations  on  the  action  of  the  specifically 
cytolytic  agencies  upon  the  egg.  It  is  probable  that  Nal  and 
NaCNS  are  slightly  soluble  in  the  egg  and  owe  their  efliciency 
to  this  fact.  That  Ca  and  bivalent  metals  in  general  inhibit 
cjd^olysis  by  bases  had  been  shown  by  the  writer  in  1906  and 
1907.1 

Herbst  made  in  1904  the  interesting  observation  that 
minute  traces  of  silver  salts  cause  a  membrane  formation  in 
the  unfertilized  sea-urchin  egg.     The  following  observation  by 

1  Loeb,  "Ueber  die  anticytolytische  Wirkung  \on  Salzen  mit  zweiwertigeu 
Metallen,"  Biochem.  Zeitschr.,  V,  351,  1907;    II,  Si,   190G. 


188     Artificial  Parthenogenesis  and  Fertilization 


him  is  especially  interesting:  ''A  new  piece  of  silver  money, 
which  had  been  specially  cleaned  before  use,  was  put  into 
26  c.c.  of  sea-water  which  contained  unfertilized  eggs  of  Echinus. 
Fifteen  minutes  later  I  noticed  in  eggs  which  lay  close  to  the 
silver  the  formation  of  membranes.  An  hour  later  membranes 
were  formed  in  some  eggs  which  were  not  so  close  to  the  silver."^ 

The  observation  by  Bohn  that  radium  may  induce  arti- 
ficial parthenogenesis  probably  belongs  in  the  same  chapter.^ 

I  believe  that  in  all  these  cases  we  are  dealing  with  cytolytic 
effects.  The  fact  shown  in  chap,  xii,  that  complete  cytolysis 
of  the  egg  by  saponin  raises  the  rate  of  oxidations  in  the  ferti- 
lized egg  to  the  same  rate  as  fertilization,  makes  it  clear  that  the 
cytolysis  of  the  surface  layer  is  the  essential  part  in  the  causation 
of  development  in  the  egg. 

9.  In  conclusion,  we  will  briefly  touch  upon  the  mechanism 
of  cytolysis.  In  the  case  of  the  chorion  of  the  eggs  of  molluscs 
and  annelids  it  can  be  directly  observed  that  substances  like 
saponin  and  benzol  exert  an  influence  that  causes  swelling  and 
liquefaction.  This  swelling,*  or  absorption  of  fluid,  and  the 
clarifying  of  cell-contents  that  were  previously  opaque,  are 
typical  of  cytolysis.  Many  authorities  follow  Koppe  in  assum- 
ing that  cj^tbiysis  depends  upon  the  solution  of  the  hypothetical 
lipoid  membrane  of  the  egg;  it  should,  however,  be  stated  that 
the  fertilization  membrane  of  the  eggs  is  insoluble  in  benzol^ 
or  in  any  other  lipoid-solvent. 

Von  Knaffl  has  developed  another  conception  of  the  mech- 
anism of  cytolysis,  which  I  shall  quote  verbatim. 

It  can  be  regarded  as  certain  that  protoplasm  is  rich  in  lipoids, 
and  that  any  chemical  or  physical  stimulus  which  produces  a  lique- 
faction or  solution  of  the  lipoids  of  protoplasm,  causes  the  egg  to  cyto- 
lyze  owing  to  the  fact  that  the  protoplasm,  being  now  free  from  hpoids, 
takes  up  water,  and  swells.  This  leads  in  many  cases  to  the  bursting 
of  the  membrane. 

1  Herbst,  Mitteil.  d.  Zool.  Station  zu  Xeapel,  XVI,  445,  1904. 
-  G.  Bohn,  Compt.  rend.  Acad.  d.  Sc,  CXXXVI,  1085,  1903. 


Membrane  Formation  and  Cytolysis  189 


The  following  conclusions  reached  by  von  Knaffl  may  be 
mentioned  by  way  of  further  explanation: 

Protoplasm  is  rich  in  hpoids;  probably  it  is  mainly  an  emulsion 
of  these  and  proteins.  Any  physical  or  chemical  stimulus  which  can 
hquefy  the  lipoids  causes  cytolysis  of  the  egg.  The  protein  of  the  egg 
can  really  only  swell  or  be  dissolved  if  the  condition  of  aggregation 
of  the  lipoid  is  altered  by  chemical  or  physical  agencies.  The  mechan- 
ism of  cjd^olysis  consists  in  the  liquefaction  of  the  lipoids,  and  there- 
upon the  lipoid-free  protein  swells  or  is  dissolved  by  taking  up  water. 
....  Hence  this  supports  Loeb's  view  that  membrane  formation  is 
induced  by  the  hquef action  of  the  hpoids. ^ 

(I  had  previously  referred  the  formation  of  the  fertilization 
membrane  to  a  liquefaction  of  the  lipoid  on  the  surface  of  the 
egg.2) 

We  will  now  examine  more  closely  the  experiments  upon 
which  von  Knaffl  based  his  view.  When  he  heated  the  sea- 
urchin  egg  to  41°  C,  cytolysis  ensued:  many  strongly  refracted 
spherules  appeared  on  the  surface  of  the  egg,  as  in  Fig.  52. 
He  regards  these  globules  as  lipoids,  since  they  disappear  or 
dissolve  in  the  presence  of  benzol,  chloroform,  and  alkali. 
But  they  do  not  disappear  in  acetone,  from  which  von  Knaffl 
concludes  that  they  consist  of  lecithin.  He  regards  the  fact 
that  in  cytolysis  the  egg  can  be  observed  to  exude  clear  drops 
which  are  soluble  in  benzol,  as  a  support  of  the  hypothesis 
that  membrane  formation  and  cytolysis  depend  essentially 
upon  lipoids  passing  into  solution,  or  being  excreted  from  the 
egg. 

Now  this  exudation  of  lipoids  may  in  reality  ex-plain  the 
clarification  of  the  egg  that  is  characteristic  of  cytolysis.  For 
if  the  protoplasm  consists  of  an  emulsion  in  which  the  walls 
of  the  vesicles  are  formed  by  a  solid  lipoid,  a  removal  of  these 
walls  must  lead  to  many  small  vesicles  flowing  together  into 

1  Von  Knaffl,  op.  cit. 

2  Loeb,    "Ueber    den    chemischen    Charakter    des    llefruchtungsvorgangs." 
Roux's  Abhandlumjeu  and   Vortrojje,  ljV\])/Aii.  1908. 


190     Artificial  Parthenogenesis  and  Fertilization 

larger  ones.  In  that  case,  less  of  the  light  passing  through  it 
will  be  reflected,  and  the  eggs  appear  clear.  A  glance  at  Fig.  51 
shows  that  as  a  matter  of  fact  the  cytolyzed  egg  consists  of 
large  drops.  It  appears  possible  that  the  outflow  of  the  clear 
drops  in  the  cj-tolysis  of  the  moderately  unpigmented  egg  of 
S.  purpuratits  may  correspond  to  the  outflow  of  haemoglobin 
from  the  red  blood  corpuscles.  It  is  to  be  noted  that  in  this 
case  it  is  unnecessary  to  assume  a  bursting  of  the  surface  layer 
of  the  red  blood  corpuscle. 

But  from  the  point  of  view  of  our  work,  it  is  not  necessary 
to  venture  upon  a  decision  with  regard  to  the  lipoid  controversy 
and  cytolj^sis.  We  can  observe  directly  that  cytolytic  sub- 
stances cause  the  chorion  that  surrounds  the  eggs  to  swell  and 
then  gradually  to  dissolve  completely.  It  is  necessary  only  for 
us  to  make  use  of  this  observation  in  order  to  understand  mem- 
brane formation  also.  If  we  assume  that  in  the  cytoplasm  of 
the  egg  itself  there  is  present  near  the  surface  a  substance  that 
is  identical  with  or  closely  allied  to  one  of  those  in  the  chorion, 
we  can  understand  that  this  must  be  made  to  swell  and  liquefy 
before  the  egg  can  develop. 

The  swelling  and  solution  of  such  a  substance  in  the  interior 
of  the  egg  causes  cytolysis,  and  hence  it  comes  about  that  all 
cytolytic  substances  or  reagents  also  produce  an  effect  that 
leads  to  membrane  formation,  and  hence  to  artificial  partheno- 
genesis. 


XVIII 

THE   FERTILIZING    EFFECT   OF    FOREIGN    BLOOD    AND 

FOREIGN  CELL  EXTRACTS 

1.  The  following  chapter  contains,  perhaps,  the  most  sur- 
prising facts  in  the  field  of  artificial  parthenogenesis.  The 
writer  succeeded  in  1907  and  1908  in  showing  that  the  blood  and 
tissue  extracts  of  many  foreign  species  will  cause  the  unfertilized 
egg  of  the  sea-urchin  (and  other  forms)  to  form  a  fertilization 
membrane  (and  develop  if  subsequently  treated  with  a  hyper- 
tonic solution),  while  the  blood  and  tissue  extracts  of  their 
own  species  have  no  such  effect.^  The  first  observation  on  the 
fertilizing  effect  of  foreign  blood  was  made  by  the  writer  in 
1907  when  he  succeeded  in  causing  membrane  formation  in 
unfertilized  sea-urchin  eggs  with  the  blood  serum  of  certain 
worms,  the  Gephyrea.     These  eggs  also  developed. 

By  making  an  incision  into  the  body  of  a  sipunculid — 
Dendrostoma  was  the  form  chiefly  used — the  fluid  of  the  body 
cavity  can  be  obtained.  This  fluid  contains  numerous  blood 
corpuscles  and  reproductive  cells,  spermatozoa  or  eggs.  In 
our  experiments  we  used  as  a  rule  only  the  body-contents  of 
female  worms.  One  c.c.  of  this  fluid  was  generalh'  diluted  with 
some  50  to  200  c.c.  of  sea-water.  The  solution  was  then  com- 
pletel}^  cleared  by  centrifuging,  and  only  tlie  perfectly  limpid 
serum,  free  from  all  solid  and  formed  constituents,  was  used  in 
our  experiments.  If,  now,  the  unfertilized  eggs  of  a  female  (sea- 
urchin)  were  put  in  a  watch  glass  with  about  3  c.c.  of  sea-water, 
and  1  to  4  drops  of  this  diluted  transparent  body-cavity  fluid 

1  Loeb,  "Ueber  die  Hervorrufung  der  Membranbildimg  beim  Seeigelci  durch 
das  Blut  gewisser  Wurraer  (Sipuiiculiden),"  P/luyer's  Archiv,  CXVIII.  36.  1907; 
"Ueber  die  Hervorrufung  der  Membraiihildung  und  Entwicklung  lieim  Seeigelei 
durcli  das  Blutserum  des  Kaninchens,  "  J^jliiger's  Arcliiv,  CXXII.  190.  1908; 
"Weitere  Versuche  ueber  die  Entwicklungserregung  des  Seeigeleies  durch  das 
Blutserum  von  Saugetieren,"   Pfluger's  Archiv,  CXXIV,  37,   190S. 

191 


192     Artificial  Parthenogenesis  and  Fertilization 


of  Dendrostoina  added,  a  certain  percentage  of  the  eggs  formed  a 
t^'pical  fertilization  membrane.  On  watching  these  eggs,  it 
was  found  that  two  to  three  hours  after  membrane  formation 
they  formed  a  normal  nuclear  spindle  and  that  some  of  them 
divided  quite  regularly  into  two  cells.  In  the  majority,  the 
next  division  did  not  take  place,  but  later  they  split  up  into 
several  cells  at  once.  Normal  appearing  eight-  and  sixteen-cell 
stages  were  very  abundant,  and  I  expected  at  first  that  the  eggs 
would  develop  into  larvae;  but  this  was  not  the  case,  with  few 
exceptions.  On  the  second  day,  most  of  the  eggs  disintegrated. 
A  few  lived  a  little  longer,  but  did  not  develop  into  blastulae. 

If  such  eggs  were  placed  immediately  after  membrane  forma- 
tion in  hypertonic  sea-water  (50  c.c.  of  sea-water+8  c.c.  2i  m 
NaCl),  all  or  most  of  the  eggs  developed  into  larvae.  If  the 
length  of  exposure  was  correctly  chosen  the  eggs  segmented  and 
developed  into  plutei  in  quite  a  normal  manner.  If  the  expo- 
sure was  too  short  the  hypertonic  solution  had  no  effect. 

It  was  not,  however,  the  eggs  of  every  sea-urchin  that  formed 
membranes  in  Dendrostoma  serum,  the  reaction  being  limited 
to  the  eggs  of  about  20  per  cent  of  the  females. 

The  next  thing  was  to  determine  more  closely  the  nature  of 
the  active  substance.  If  the  reactions  of  the  effective  solution 
of  Dendrostoma  blood  are  tested,  it  will  be  found  that  it  reacts 
to  neutral  red  just  like  ordinary  sea-water.  This  excludes  the 
possibility  of  membrane  formation  being  due  to  one  of  the 
lower  fatty  acids  (or  any  other  acid).  For  in  order  to  cause 
membrane  formation  by  means  of  one  of  the  lower  fatty  acids, 
about  3  c.c.  of  N/10  acid  must  be  added  to  50  c.c.~oi  sea-water, 
and  this  renders  the  latter  strongly  acid.  Moreover,  the. eggs 
must  not  remain  in  this  solution  longer  than  1 J  to  2 J  minutes  (at 
15°  C),  else  no  membrane  is  formed.  In  the  third  place,  the 
membrane  is  never  formed  in  the  acid  sea-water  (in  the  case  of 
the  lower  fatty  acids),  but  only  after  the  egg  has  been  trans- 
ferred to  ordinary  (i.e.,  faintly  alkaline)  sea-water.     But  when 


Effect  of  Foreign  Blood  and  Cell  Extracts         193 

the  egg  is  exposed  to  the  diluted  sipuncuHd  blood,  membrane 
formation  takes  place  in  the  presence  of  the  blood. 

However,  it  is  also  improbable  that  it  is  due  to  any  of 
the  hydrocarbons  such  as  benzol,  toluol,  and  amylene,  or  to 
saponin  or  any  similar  glucoside.  For  all  these  substances 
lead,  not  only  to  membrane  formation,  but  also  to  cytolysis 
of  the  egg,  if  it  is  not  removed  from  the  solution  immediately 
after  membrane  formation.  But  the  sipunculid  serum  does  not 
cytolyze  the  egg,  at  least  not  in  the  concentration  necessary  to 
produce  membrane  formation. 

The  question  of  the  thermostability  of  the  effective  con- 
stituent of  the  sipunculid  serum  was  next  taken  into  considera- 
tion. In  order  to  avoid  the  suspicion  of  infection  with  living 
spermatozoa,  it  had  already  been  found  necessary  in  these 
experiments  to  heat  the  sipunculid  serum  to  between  50°  and 
60°  C.  Prolonged  heating  at  60°  C.  does  not  decrease  the 
effectiveness  of  the  serum,  nor  does  heating  it  to  70°  or  80°  C. 
vSuddenly  heating  the  serum  to  boiling-point  in  one  case  reduced 
its  efficacy  to  one-third  of  its  original  amount.  Prolonged  boil- 
ing (two  to  three  minutes)  has  completely  destroyed  the  efficacy 
in  all  the  cases  hitherto  observed. 

Even  when  the  blood  was  heated  for  an  hour  to  60°  C.  it 
did  not  lose  its  effect. 

It  was  subsequently  found  that  the  blood  and  tissue  extracts 
of  a  large  number  of  animals. had  a  similar  effect,  although 
they  did  not  act  in  such  a  high  degree  of  dilution.  The  most 
welcome  observation  was  that  the  blood  of  mannnals  (rab])it, 
pig,  ox,  etc.)  was  very  active,  since  this  enabled  us  to  under- 
take a  more  systematic  investigation  of  this  field.  The  writer 
succeeded  in  producing  membrane  formation  in  sea-urchin  eggs 
with  the  blood  of  mammals  (dog,  pig,  and  ox).  The  serum 
was  rendered  isotonic  with  soa-wator  by  th(^  addition  of  a 
2J  m  NaCl  solution  (1  c.c.  of  the  2^  m  NaCl  solution  was 
added  to  6.5  c.c.  of  the  serum). 


194     Artificial  Parthenogenesis  and  Fertilization 

It  was  observed  in  the  experiments  with  the  blood  of 
mammals  that,  just  as  with  the  Dendrostoma  blood,  it  was  not 
the  eggs  of  every  sea-urchin  that  would  respond,  but  only  the 
eggs  of  about  10  per  cent  of  the  females.  I  am  inclined  to 
attribute  this  to  differences  in  the  permeability  of  the  eggs  of 
different  females.  In  order  that  the  blood  may  cause  membrane 
formation,  it  is  necessary  for  its  effective  constituent  to  diffuse 
into  the  egg.  It  seems  then  that  the  necessary  degree  of  per- 
meability will  not  be  found  in  the  eggs  of  every  female,  but 
only  in  those  of  a  certain  percentage.  Moreover,  the  blood  of 
mammals  is  less  effective  than  that  of  Dendrostoma.  Whereas 
the  latter  produces  membrane  formation  when  diluted  100  to 
1,000  times,  the  former  is  effective  only  in  2  to  10  times  dilution.^ 
The  experiments  succeed  best  when  the  eggs  are  taken  fresh 
from  the  ovary. 

2.  The  fact  that  not  the  eggs  of  every  female  reacted  with 
foreign  blood  made  it  necessary  to  find  methods  of  sensitizing 
the  eggs  to  the  effects  of  foreign  blood.  Various  methods  were 
tried.  A  rise  in  temperature  seemed  at  first  promising.  In  the 
following  experiment  the  eggs  of  a  female  were  used  of  which 
about  3  per  cent  formed  membranes  with  ox  serum  at  room 
temperature. 

The  eggs  of  this  female  were  put  in  a  beaker  wdth  sea-water; 
the  ox  serum  was  put  in  a  second  beaker,  and  both  were  heated 
slowly  in  a  water  bath.  At  certain  temperatures  0.5  c.c.  of 
sea-water + eggs  and  0.5  c.c.  of  serum  were  mixed  in  a  watch 
glass,  and  the  percentage  of  eggs  that  formed  membranes 
was  estimated.  The  result  of  one  such  experiment  is  given  in 
Table  XXXVI. 

1  T.  B.  Robertson  has  recently  found  that  ox  serum  acts  best  in  a  dilution  of 
1 :  16  with  sea-water.  He  even  obtained  results  with  a  greater  dilution  (Robertson, 
Archiv  /.  Entwicklungsmechanik,  XXXV,  70,  1912).  Wasteneys  and  I  did  not 
notice  such  an  effect  of  dilution  on  the  egg  of  Arbacia,  where  the  results  agreed 
with  the  writer's  former  observations  on  S.  -purpuratus.  Robertson  ascribes 
the  beneficial  effect  of  dilution  to  the  presence  of  an  inhibiting  factor  in  the 
serum. 


Effect  of  Foreign  Blood  and  Cell  Extracts     195 


It  will  be  seen  that  when  the  eggs  are  warmed  to  31°  C. 
a  sudden  increase  in  the  number  of  membrane  formations 
occurs.  At  36°  this  effect  lapses  again  owing  to  the  modifica- 
tion of  the  process  of  membrane  formation.     For  clear  drops 

TABLE  XXXVI 


Temperature 

Percentage  of 
Eggs  That 

Formed 
Membranes 

Temperature 

Percentage  of 
Eggs  That 

Formed 
Membranes 

15°  C                   

3 
3 
5 

70 

32°  C 

100 

28°  C            

34°  C 

100 

30°  C               

36°  C 

1 

31°  C    

37°  C 

0 

exude  from  the  egg,  but  their  surfaces  do  not  flow  together  so 
as  to  produce  a  uniform  fertilization  membrane.  Membrane 
formation  does  not  occur  until  the  eggs  are  cooled  again. 
Merely  heating  the  eggs  to  32°  C.  without  the  addition  of 
serum  does  not  lead  to  membrane  formation.  If  the  eggs  of 
S.  purpuratus  are  heated  to  31°  or  over,  their  capacity  for 
development  is  destroyed. 

But  I  discovered  another  method  of  increasing  the  efficacy 
of  the  blood  serum  without  reducing  the  capacity  of  the  eggs 
for  development.  This  consists  in  the  addition  of  a  m  ^2  SrCl, 
solution  to  the  serum. 

In  order  to  demonstrate  the  beneficial  eflfect  of  strontium, 
one  must  choose  eggs  that  show  only  a  slight  degree  of  sensi- 
tiveness toward  serum.  Table  XXXVII  sums  up  the  influence 
of  strontium  upon  the  number  of  eggs  which  can  be  made  to 
develop  by  serum.  Each  experiment  was  carried  out  upon  tlie 
eggs  of  a  single  female. 

In  all  these  experiments  I  employed  a  3/8  grannnolecular 
solution  of  SrCls.  Somewhat  better  results  were  obtained  if 
about  six  drops  of  sea-water  were  added  to  the  mixture  of  scTum 
and  SrCU.  The  addition  of  BaClg  had  a  similar  effect  in  tliat 
of  SrClg.     But  it  is  troublesome  to  work  with  Bad.,  on  account 


196     Artificial  Parthenogenesis  and  Fertilization 


of  the  heavy  precipitate  of  BaS04  that  is  formed  in  this  case, 
hence  I  have  not  performed  so  many  experiments  with  barium. 
Curiously  enough  Mg  and  Ca  do  not  help  the  formation  of 
a  membrane  with  mammalian  blood. 

TABLE  XXXVII 


Composition  of  the  Solution 

Percentage 
of  Eggs 

That 

Formed 

Membranes 

^     /2  c.c.  sea-water  +  1  drop  ox  serum. 

\2  c.c.  SrCL+l  drop  ox  serum 

2    |2  c.c.  sea- water +3  drops  ox  serum . 

i2  c.c.  SrCl2+3  drops  ox  serum  .... 

o     [1  c.c.  sea-water+3  c.c.  ox  serum.  . 

\l  c.c.  SrCh  +  l  c.c.  ox  serum 

A    /I  c.c.  sea-water  +  1  c.c.  ox  serum.  . 

'  \1  c.c.  SrCU  +  l  c.c.  ox  serum 

c    /I  c.c.  sea-water  +  1  c.c.  ox  serum.  . 

'  \1  c.c.  SrCL  +  l  c.c.  ox  serum 

r.    /I  c.c.  sea-water  +  1  c.c.  pig  serum.. 

■  \1  c.c.  SrCL  +  l  c.c.  pig  serum 

y    /I  c.c.  sea-water  +  1  c.c.  pig  serum.. 

\1  c.c.  SrCla  +  l  c.c.  pig  serum 

r,    /2  c.c.  sea-water +2  c.c.  dog  serum. 
\2  c.c.  SrCl2+2  c.c.  dog  serum 

5 
80 

0 
80 
14 
33 

5 
26 

8 
50 

5 
80 

** 

0 

40 

0 

50 

The  efficacy  of  Dendrostoma  blood,  however,  was  not  in- 
creased by  the  addition  of  strontium. 

3.  This  method  of  sensitizing  was  improved  by  putting  the 
eggs  for  some  time  into  a  3/8  m  solution  of  SrCl2  and  then 
exposing  them  to  the  serum. ^  The  eggs  are  first  washed  in 
a  NaCl  solution  and  then  put  into  a  3/8  m  solution  of  SrCl2- 
After  from  five  to  ten  minutes  (or  if  necessary  later)  a  drop  of 
eggs  is  taken  from  this  solution  and  put  into  a  mixture  of  1  c.c. 
sea-water  and  1  c.c.  sermn  (rendered  isosmotic  with  sea-water). 
In  this  way  by  a  sufficiently  long  treatment  with  SrCl2  the  eggs 
of  practically  every  female  can  be  sensitized  against  foreign 
blood.     The  same  method  was  found  effective  for  the  eggs  of 

1  Loeb,  "Die  Sensitivierung  der  Seeigeleier  mittelst  Strontiumchlorid  gegen 
die  entwicklungserregende  Wirkung  von  Zellextracten,"  Archiv  f.  Entwicklungs- 
mechanik,  XXX.  44,  1910. 


Effect  of  Foreign  Blood  and  Cell  Extracts      197 

ArbaciaJ  It  was  found  that  if  the  eggs  were  sensitized  against 
ox  serum  they  are  sensitized  against  other  foreign  blood  and 
tissue  extracts. 

The  question  is :  How  does  SrCls  (or  BaClg)  cause  these  sensi- 
tizing effects?  This  is  possibly  answered  by  the  observation 
that  if  the  unfertilized  eggs  of  purpuratus  remain  permanently' 
in  the  SrClj  solution,  they  will  ultimately  form  membranes 
without  requiring  any  further  treatment.  The  time  required 
for  this  effect  differs  for  the  eggs  of  different  females.  If  eggs 
which  have  formed  a  membrane  upon  treatment  with  SrCU 
(or  BaClg)  are  subsequently  exposed  to  a  hypertonic  solution 
they  will  develop  into  larvae.  The  fact  that  the  SrClg  alone 
can  cause  membrane  formation  if  the  eggs  are  exposed  to  it 
for  a  sufficiently  long  time  suggests  that  the  sensitivation  con- 
sists in  a  modification  of  the  cortical  layer  of  the  egg  of  a  char- 
acter similar  to  that  which  leads  to  membrane  formation.  SrCU 
thus  facilitates  the  subsequent  membrane  formation  by  serum. 

The  following  facts  are  of  interest.  I  had  already  noticed 
in  my  experiments  on  heterogeneous  h>^bridization  that  the  eo-o-s 
of  the  sea-urchin  can  be  fertilized  in  larger  numbers  with  the 
sperm  of  Asterias  than  with  the  sperm  of  Pycnopodia  or  Asterma. 
It  was  found  that  the  extracts  of  the  coccum  of  these  three 
species  of  starfish  showed  the  same  relative  difference  in  their 
power  of  causing  membrane  formations  in  the  unfertilized 
egg  of  S.  purpuratus  that  had  previously  been  sensitized  with 
SrClg.  This  agrees  with  the  fact,  which  we  shall  prove  in  the 
next,  chapter,  that  the  membrane  formation  by  the  spermato- 
zoon is  caused  also  by  a  cytolytic  agent—a  lysin;  and  that  the 
lysins  contained  in  the  coecum  show  the  same  relative  efliciencv 
as  the  lysins  contained  in  the  spermatozoa  of  the  three  species. 
It  may  be  of  interest  that  the  extracts  of  all  kinds  of  starfish 
cells,  even  of  the  eggs,  were  able  to  bring  about  the  membrane 
formation  in  the  sea-urchin  egg. 

»  Loeb  and  Wasteneys,  Science,  XXXVI,  255.  1912. 


198     Artificial  Parthenogenesis  and  Fertilization 


While  it  was  thus  easy  to  cause  the  membrane  formation  of 
the  unfertiUzed  sea-urchin  egg  with  blood  or  extracts  of  tissues 
from  foreign  species,  it  was  almost  impossible  to  bring  about 
the  membrane  formation  in  the  sea-urchin  egg  with  extracts 
from  the  tissues  of  the  sea-urchins.  Only  in  one  among  many 
attempts  did  the  writer  succeed  in  causing  membrane  formation 
in  the  eggs  of  *S'.  purpiiratus  by  the  watery  extract  from  the 
coecum  of  S.  franciscanus.  The  extract  had  been  standing  five 
days;  newly  prepared  extract  was  without  effect  upon  the  same 
eggs.     About  5  per  cent  of  the  eggs  formed  membranes. 

We  may  ask  the  question  why  it  is  that  blood  or  the  extracts 
of  tissues  of  foreign  species  will  readily  cause  membrane  forma- 
tion in  the  unfertilized  eggs  of  sea-urchins,  while  the  extract 
from  tissues  of  the  sea-urchin  remains  ineffective.  From  the 
experiments  with  acids  and  alkah  it  became  evident  that  a 
necessary  prerequisite  of  the  efficiency  of  a  substance  for  the 
causation  of  membrane  formation  is  its  diffusion  into  the  egg, 
or  at  least  into  its  cortical  layer.  The  same  can  be  shown  to 
be  true  for  the  hydrocarbons,  ether,  alcohols,  and  the  other 
substances  which  cause  membrane  formation.  It  is  therefore 
possible  that  the  inefficiency  of  the  blood  and  tissue  extracts 
of  the  same  animal  and  the  efficiency  of  the  blood  of  foreign 
species  for  the  causation  of  membrane  formation  is  due  to  the 
fact  that  the  foreign  lysins  can  diffuse  into  the  egg  while  the 
lysins  of  the  same  species  cannot. 

We  know  that  the  blood  of  all  animals  carries  lysins  which 
may  cause  haemolysis  in  foreign  forms,  but  are  harmless  to 
the  cells  of  the  animal  itself.  What  causes  this  immunity  ? 
According  to  our  investigations  the  immunity  of  our  cells 
against  the  lysins  contained  in  our  blood  may  be  due  to  the 
fact  that  the  lysins  of  our  blood  cannot  diffuse  into  our  own 
cells  while  they  may  diffuse  into  the  cells  of  foreign  species. 

This  view  is  supported  by  some  data  which  will  be  given  in 
the  next  chapter  and  to  which  we  may  refer  briefly  here.     The 


Effect  of  Foreign  Blood  and  Cell  Extracts      199 

watery  extract  of  spermatozoa  of  foreign  species  causes  mem- 
brane formation  in  the  sea-urchin  egg.  The  watery  extract  of 
the  spermatozoa  of  the  sea-urchin  does  not  produce  membrane 
formation  in  the  sea-urchin  egg.  We  cannot  say  that  this  is 
due  to  the  fact  that  the  sea-urchin  sperm  contains  no  memljrane- 
forming  substance.  The  only  possible  conclusion  is  the  para- 
doxical assumption  that  the  membrane-forming  substance  of 
many  foreign  spermatozoa  can  diffuse  into  the  sea-urchin  egg, 
while  the  membrane-forming  substance  of  the  spermatozoon  of 
its  own  species  cannot  get  into  the  egg  by  diffusion,  but  must 
be  carried  into  it  by  the  living  spermatozoon. 

4.  Experiments  on  the  isolation  of  the  substance  present 
in  the  serum  which  is  responsible  for  membrane  formation  have 
not  yet  met  with  much  success.  It  is  apparent  that  this  sub- 
stance is  comparatively  resistant  to  heat. 

For  example,  ox  serum  was  slowly  heated  on  the  water  bath 
and  0.5  c.c.  removed  at  different  temperatures  and  put  into 
watch  glasses.  On  cooling,  0.5  c.c.  of  sea-water  was  added  and 
a  drop  of  eggs  placed  in  the  mixture.  It  was  found  that  the 
activity  of  the  serum  was  not  impaired  by  heating  it  up  to 
73°  C.  I  took  as  criterion  the  number  of  eggs  that  formed 
membranes  in  the  solution.  The  serum  coagulated  at  73°  C. 
On  heating  the  coagulum  to  100°,  the  clear  liquid  that  could 
be  squeezed  out  had  no  longer  any  effect  on  the  eggs. 

The  activity  of  the  serum  is  apparently  not  decreased  by 
putrefaction;  nor  does  extraction  of  the  serum  with  ether, 
four  times  repeated,  lessen  its  effect.  But  as  ether  can  cause 
membrane  formation  and  cytolysis,  one  must  take  the  precau- 
tion in  these  experiments  of  not  using  the  serum  until  all  the 
ether  has  been  expelled. 

If  a  large  quantity  of  acetone  is  added  to  the  ox  serum,  a 
voluminous  precipitate  is  obtained,  which  hardens  on  drying 
into  a  brown  crust.  This  substance  is  insoluble  in  sea-water. 
This  dried  acetone  precipitate  was  ground  up  in  sea-water,  in  a 


200     Artificial  Parthenogenesis  and  Fertilization 

mortar,  filtered,  and  the  filtrate  tested  for  its  action.  This 
filtrate  is  exceptionally  active.  • 

Robertson  described  a  more  complicated  process  of  precipi- 
tating the  active  substance  of  the  blood  with  acetone,  which 
was  based  on  the  assumption  ''that  the  fertilizing  agent  might 
be  precipitable  by  alkaline  earths."^ 

He  has  since  improved  this  method  and  gives  the  follow- 
ing description:  Precipitation  from  the  serum  by  acetone, 
extraction  of  the  precipitate  with  hot  N/10  HCl,  exactly 
neutrahzing  the  extract  with  Ba(0H)2,  redissolving  the  precipi- 
tate in  N/10  H2SO4  and  reprecipitating  it  with  acetone.  The 
yield  from  a  liter  of  ox  serum  lies  between  10  and  40  milligrams. 
From  its  reactions  Robertson  concludes  that  the  substance  is 
either  a  protein  or  a  peptone.  One  part  of  the  substance  rubbed 
up  in  512,000  parts  of  sea-water  caused  membrane  formation 
in  80  per  cent  of  the  eggs  of  S.  purpuratus  which  had  previously 
been  sensitized  by  four  minutes'  immersion  in  3/8  m  SrCl2. 

Robertson  also  found  that  Witte's  peptone  contains  a  mem- 
brane-forming substance  demonstrable  by  eggs  previously 
treated  with  SrCl2.  He  thinks  it  unlikely  that  the  active  sub- 
stance is  a  lipoid. 2 

1  Robertson,   Archiv  f.   Entwicklungsmechanik,  XXXV,   70,   1912. 

-  Robertson,    Proc.   Society  for   Exper.   Biol,  and  Med.,   X,    117,    1913. 


XIX 

THE  FERTILIZING  EFFECT  OF  SPERM  EXTRACT 

1.  In  1899  Pieri  published  a  note  to  the  effect  that  by  merely 
shaking  up  the  testes  of  the  sea-urchin  in  sea-water  he  had 
been  able  to  extract  a  substance  which  fertilized  the  egg  of  the 
sea-urchin.^  The  sea- water  containing  the  spermatozoa  was 
filtered  after  the  shaking,  and  the  filtrate  added  to  the  eggs. 
The  eggs  developed.  As  the  author  himself  states,  and  as  is 
generally  known,  spermatozoa  pass  through  filter  paper,  and 
so  one  cannot  quite  understand  on  what  the  author  bases  his 
statement  that  we  are  concerned  here  with  a  fertilization  by 
sperm  extract,  and  not  by  living  spermatozoa;  control  experi- 
ments were  not  performed.  A  better  addition  to  the  solution 
of  the  problem  was  made  by  Winkler.^  He  states  that  his 
work  has  not  gone  beyond  a  preliminary  stage.  His  experi- 
ments consisted  in  putting  the  spermatozoa  of  two  kinds  of 
Naples  sea-urchins,  Sphaerechinus  and  Arbacia,  into  distilled 
w^ater  for  half  an  hour.  The  filtrate  was  able  to  produce  the 
first  segmentation  stages  in  the  sea-urchin.  It  will  be  seen, 
however,  that  Winkler  did  not  work  with  unaltered  sea-water, 
and  it  is  possible  that  the  alteration  of  the  sea-water  and  not 
the  hypothetical  substances  extracted  from  the  sperm  was  the 
cause  of  the  segmentation  that  he  observed. 

If  the  spermatozoa  were  simply  killed  by  being  heated  in  sea- 
water  to  between  50°  and  60°  C,  and  the  eggs  put  in  this  liquid, 
nothing  happened.     But  if  they  were  put  into  distilled  water  for  half 

1  T.  B.  Pieri.  "  Un  nouveau  ferment  soluble:  L'ovulase,"  Arch.de  Zool.  exper. 
etgen.,  XXIX,    1899. 

-  H.  Winkler,  "  Ueber  die  Furcliung  unbefruchteter  Eier  unter  der  Einwirkung 
von  Extractiv'stofTen  aus  deni  Sperma,"  Nachrichten  der  Ges.  d.  Wissenach.  zu 
Gottingen,  1900.   187. 

201 


202    Artificial  Parthenogenesis  and  Fertilization 


an  hour  and  frequently  shaken  up,  the  fluid  then  proved  effective. 
Of  course,  I  did  not  use  it  directly,  but  first  filtered  it  five  to  six  times 
through  triple  filter  paper,  and  then  added  the  salts  obtained  by  the 
evaporation  of  sea-water  so  as  to  make  it  of  the  same  concentration 
as  that  of  normal  sea- water  (about  4  per  cent).  When  put  into  this 
water,  unfertilized  eggs  of  both  Sphaerechinus  and  Arbacia  showed 
signs  of  cleavage — each,  of  course,  only  responding  to  the  extract  of 
sperm  of  the  same  species.  I  may,  however,  remark  at  the  same  time 
that  not  a  very  large  number  and  by  no  means  all  of  the  eggs  responded; 
it  was  usually  the  case  that  the  eggs  of  one  individual  would  react  to 
one  and  the  same  sperm  fluid,  but  not  those  of  another.  In  the  best 
cases  the  segmentation  proceeded  regularly  to  the  four-cell  stage,  but 
afterward  it  became  abnormal  and  the  blastomeres  which  were  very 
dissimilar  in  size  fell  apart  from  one  another,  probably  as  the  result  of 
the  absence  of  the  vitelHne  membrane.  The  velocity  of  development 
was  much  slower  in  eggs  so  treated  than  in  normally  fertilized  ones. 

I  have  no  intention  of  criticizing  Winkler's  efforts;  they 
were  certainly  a  step  in  the  right  direction.  But  it  must  be 
pointed  out  that  the  experiments  are  not  free  from  objection. 
In  the  first  place,  his  sea-water  was  made  much  more  alkaline 
than  is  normal,  owing  to  the  fact  that  by  first  evaporating  it 
he  drove  out  the  CO2  and  converted  the  bicarbonate  into  car- 
bonate. He  afterward  restored  the  sea-water  to  its  normal  vol- 
ume by  adding  distilled  water.  The  increase  of  alkalinity  thus 
produced  will  alone  lead  to  such  results  as  Winkler  describes, 
as  I  show^ed  years  ago. 

For  several  years  I  have  tried  in  vain  to  repeat  Winkler's 
experiments;  results  such  as  he  described  can  be  obtained 
by  using  slightly  hypertonic  or  hyperalkaline  sea-water,  or 
the  two  together;  but  these  results  are  also  obtained  when  no 
sperm  is  added  to  such  sea-water.  Before  the  appearance  of 
Winkler's  paper  I  myself  had  examined  the  effect  of  various 
enzymes  upon  the  unfertilized  sea-urchin  egg,  but  with  negative 
results.  At  my  suggestion,  Professor  W.  J.  Gies,  of  Columbia 
University,  undertook  a  series  of  experiments  in  which  he 
subjected  the  spermatozoa  to  every  known  method  that  leads 


Effect  of  Sperm  Extract  203 


to  the  extraction  of  enzymes  from  cells. ^  Extracts  of  spermato- 
zoa in  fresh  water,  sea-water,  alcohol,  ether,  glycerin,  alkalies- 
all  proved  absolutely  ineffective.  The  repetition  of  Winkler's 
experiments  with  special  attention  to  the  sources  of  error  led  to 
negative  results.  Gies  concluded  from  these  experiments  that 
if  the  spermatozoon  does  cause  the  development  of  the  egg  by 
means  of  an  enzyme,  that  enzyme  either  cannot  be  extracted 
from  the  sperm  by  the  usual  methods,  or  it  is  unable  to  enter 
the  egg.  It  would  also  be  difficult  to  understand  why  Winkler 
observed  no  membrane  formation  if  the  development  really 
depended  upon  a  sperm  extract. 

2.  Kupelwieser  found  in  the  writer's  laboratory  at  Pacific 
Grove  that  if  the  eggs  of  the  sea-urchin  are  placed  in  very  con- 
centrated Mytilus  sperm  they  form  a  typical  fertilization  mem- 
brane in  from  five  to  fifteen  minutes.  The  eggs  behave  just 
like  those  in  which  artificial  membrane  formation  has  been 
produced  by  a  fatty  acid.  They  develop  only  when  subse- 
quently exposed  to  hypertonic  sea-water,  otherwise  they  dis- 
integrate. The  rapid  formation  of  the  fertilization  membrane 
in  this  case  obviously  bars  the  entrance  of  the  spermatozoon 
into  the  egg,  a  process  that  goes  on  much  more  slowly  than 
membrane  formation.  Kupelwieser  succeeded  subsequently  in 
obtaining  similar  results  with  the  filtrate  from  spermatozoa 
that  had  been  previously  killed  by  heating  to  between  70° 
and  100°. 

I  then  tried  the  filtered  sperm  of  Chiton,  Asterias,  Asterina, 
SJranciscanus  and  purpuratus,  all  of  which  had  been  heated  to  between 
70°  and  100°,  In  all  cases  I  obtained  membrane  formation  (with  the 
eggs  of  S.  purpuratus). 

The  fundamental  point  about  this  membrane  formation  was 

that  the  concentration  of  the  sperm  must  be  as  high  as  possible. 

It  is  best  to  place  the  eggs  directly  in  the  living  t>pcrm  with  little 

or  no  dilution,  for  comparison  with  the  dead  and  filtered  sperm  that 

»  W.  J.  Gies,  "Do  Spermatozoa  Contain  an  Enzyme.  Having  the  Power  of 
Causing  the  Development  of  Mature  Ova  ?"    Am.  Jour.  I'hysiul.,  VI.  53.  1901. 


204     Artificial  Parthenogenesis  and  Fertilization 

has  been  diluted  at  most  with  its  own  volume  of  sea- water.  In  this 
way  I  obtained  in  some  cases  as  many  as  90  per  cent  of  membranes. 
But  I  must  particularly  point  out  the  fact  that  these  experiments  only 
succeed  with  the  eggs  of  at  most  one  female  in  five.  In  all  these  cases 
the  eggs  behave  after  membrane  formation  just  like  those  in  which 
membrane  formation  has  been  evoked  by  a  fatty  acid.^ 

3.  This  statement  of  Kupehvieser  is  correct  except  in  one 
point,  namely,  in  regard  to  the  effect  of  dead  sea-urchin  sperm 
upon  the  sea-urchin  egg.  I  have  made  many  experiments  in 
regard  to  the  effect  of  the  extract  of  sperm  upon  membrane 
formation  and  found  that  it  does  not  differ  from  the  membrane 
formation  by  extracts  of  other  tissues.  While  the  eggs  of  the 
majority  of  purpuratus  are  immune  against  the  watery  extract 
of  dead  sperm  of  foreign  species,  if  they  are  sensitized  by  a 
treatment  with  SrCl2,  all  or  part  of  the  eggs  of  practically 
every  female  will  form  a  fertilization  membrane,^  if  some  ex- 
tract of  dead  sperm  of  a  foreign  species  is  added.  The  extract 
of  dead  sea-urchin  sperm,  however,  was  in  all  cases  absolutely 
ineffective  upon  the  eggs  of  sea-urchins.  In  my  experiments 
the  spermatozoa  were  killed  by  keeping  them  for  20  minutes  at 
a  temperature  of  about  50°  C.  While  the  extract  of  foreign 
spermatozoa  killed  in  this  w^ay  was  very  efficient,  the  extract 
of  spermatozoa  of  the  sea-urchin  killed  in  the  same  way  wa& 
absolutely  without  effect  upon  the  sea-urchin  egg. 

We  have  thus  the  paradoxical  fact  that  foreign  sperm  can 
cause  membrane  formation  and  in  certain  cases  development 
of  the  sea-urchin  egg,  no  matter  whether  the  sperm  is  dead  or 
alive;  while  sperm  of  the  sea-urchin  can  bring  about  fertili- 
zation of  the  sea-urchin  egg  only  if  it  is  alive.  The  explanation 
of  this  paradox  lies  in  the  statement  given  in  the  preceding^ 
chapter  that  the  lysins  of  foreign  animals  can  get  into  the  cells 

1  H.  Kupelwieser,  "Versuche  ueber  Entwicklungserregung  und  Membran- 
bildung  bei  Seeigeleiern  durch  MoUuskensperma,"  Biol.  Centralbl.,  XXVI,  744.. 
1906;    Archiv  f.  Entwicklungsmechanik,  XXVII,  434,   1909. 

2  SrCL  does  not  increase  the  fertilizing  power  of  living  sperm. 


Effect  of  Sperm  Extract  205 


by  mere  diffusion,  while  the  lysins  of  the  same  species  cannot 
get  into  the  egg  by  diffusion.  Only  through  the  motive  power 
of  the  living  spermatozoon  w^hich  acts  as  a  carrier  can  the  ferti- 
lizing lysin  of  the  animal's  o\\ti  species  get  into  the  egg. 

If  the  eggs  were  not  immune  against  the  lysins  of  their  own 
species,  it  would  be  inevitable  that  their  development  would  be 
caused  by  the  lysins  of  the  blood  or  the  body  liquids  of  the 
female.  They  would  all  be  caused  to  begin  to  develop  and  then 
perish,  and  this  would  cause  the  extinction  of  the  species; 
or  if  a  complete  development  were  possible,  parthenogenesis 
would  be  the  rule.  This  would  lead  to  the  extinction  of  all 
forms  of  animals  in  which  the  male  is  heterozygous  for  sex, 
since  the  offspring  would  all  be  males. 

Leo  Loeb^  has  published  observations  w^hich  make  it  probable 
that  in  the  ovary  of  higher  animals  a  small  percentage  of  eggs 
can  begin  a  parthenogenetic  development.  He  found  in  about 
10  per  cent  of  the  ovaries  of  guinea-pigs  between  the  ages  of  two 
and  six  months  "transitory  tumors"  (chorion-epitheliomata) 
which  cannot  be  interpreted  in  any  other  way  than  as  young 
embryos,  which,  however,  undergo  an  abnormal  development. 
These  tumors  seem  to  originate  from  eggs  lying  in  the  super- 
ficial layer  of  the  ovary.  A  kind  of  placenta  is  formed.  It  is 
possible  that  the  embryomata  and  chorion-epitheliomata  found 
occasionally  in  human  sexual  glands  also  owe  their  origin  to  the 
beginning  of  a  parthenogenetic  development  of  eggs. 

4.  For  the  sake  of  completeness  the  following  facts  should 
be  mentioned.  When  we  add  living  spermatozoa  of  foreign 
species,  e.g.,  of  the  shark  or  even  of  starfish,  to  eggs  of  S.  pur- 
puratus  in  normal  sea-water  we  do  not,  as  a  rule,  get  a  mem- 
brane formation.  But  when  we  use  the  extract  of  dead  sperm 
of  these  species,  the  unfertilized  eggs  of  purpiiratus  may  form 
membranes  in  normal  sea-water,  especially  if  the  eggs  have 
been  previously  sensitized.     This  difference  is  accounted  for  by 

1  Leo  Loeb,  Zeitschr.  /.  Krebs/orschung,  XI,  259,  1012. 


206     Artificial  Parthenogenesis  and  Fertliization 

the  fact  that  if  we  use  extract  of  dead  sperm,  the  membrane- 
forming  substances  will  reach  the  egg  in  higher  concentrations 
than  if  a  single  spermatozoon  of  a  foreign  species  reaches  it. 
It  seems  that  a  living  spermatozoon  must  come  in  close  contact 
with  the  fertilization  cone  of  the  egg  before  membrane  forma- 
tion is  possible.  This  seems  possible  only  for  the  foreign 
spermatozoon  if  we  raise  the  alkalinity  of  the  sea-water  through 
the  addition  of  some  NaOH. 

When  we  use  the  eggs  of  another  sea-urchin,  S.  franciscanus, 
the  result  is  entirely  different.  Even  the  living  spermatozoa  of 
starfish  or  the  shark,  or  even  of  warm-blooded  animals  like  the 
fowl,  bring  about  the  membrane  formation  in  this  egg  in 
normal  sea-water.  But  no  egg  develops;  they  all  behave  in 
this  case  as  if  only  the  membrane  formation  with  butyric  acid 
had  been  called  forth.  We  see  from  this  that  we  cannot  expect 
that  all  species  of  sea-urchins  behave  alike  in  every  respect. 

T.  B.  Robertson  has  worked  out  a  method  which  permits 
the  extraction  of  a  substance  from  the  testicles  of  the  sea- 
urchin  which  produces  membrane  formation  in  the  sea-urchin 
egg.i 

IT.  B.  Robertson,  Archiv  f.  Entwicklungsmechanih,  XXXV,  64,  1912. 


XX 

THE  MECHANISM  OF  THE  FORMATION  OF  THE 
FERTILIZATION  MEMBRANE^ 

1.  We  can  safely  state  that  the  previous  experiments  have 
all  clearly  demonstrated  one  fact :  that  the  initiation  of  develop- 
ment in  the  sea-urchin  egg  is  due  to  a  change  in  the  surface  of 
the  egg — apparently  a  cytolysis  of  the  cortical  layer — which 
results  generally  in  a  membrane  formation.  In  some  cases 
this  membrane  is  more  typically  developed  than  in  others.  We 
shall  now  communicate  some  experiments  concerning  the  nature 
of  this  process. 

The  reader  remembers  from  chap,  ii,  that  if  the  mem- 
brane formation  proceeds  slowly  in  the  egg  of  S.  purpuratus, 
it  begins  with  the  formation  of  minute  blisters  at  the  surface 
of  the  egg.  These  blisters  grow  in  size  and  their  contents  fuse 
while  the  surface  film  of  all  the  blisters  forms  the  outer  fertiliza- 
tion membrane.  This  membrane  is  separated  by  the  fluid  (the 
fused  contents  of  the  individual  blisters)  from  the  protoplasm 
of  the  egg.  What  is  the  origin  of  this  fluid  ?  Is  it  secreted  by 
the  egg,  or  is  it  absorbed  from  the  sea-water?  If  it  were 
secreted  entirely  from  the  egg,  the  diameter  of  the  cytoplasm 
should  show^  a  decrease  after  membrane  formation.  However, 
measurements  that  I  have  made  showed  that  the  egg  c>i;oplasm 
undergoes  no  remarkable  diminution  of  its  volume  at  membrane 
formation.  Hence  the  essential  part  of  the  fluid  that  lies  between 
the  cytoplasm  and  the  membrane  must  be  derived  from  without, 
i.e.,  from  the  sea-water.  But  one  part  is  derived  from  the  egg, 
and  this  latter  part  is,  as  we  shall  see,  a  colloid. 

1  Loeb,  "Uebcr  die  osmotischen  Eigenschaften  und  die  Entstohung  dor 
Befruchtimgsmembran  beim  Seeigelei,"  Archiv  f.  Entwicklungsmechanik,  XXVI. 
82.  1908. 

207 


208     Artificial  Parthenogenesis  and  Fertilization 


2.  The  existence  and  role  of  this  colloid  becomes  clear 
through  the  following  experiments.  When  we  put  fertilized 
eggs  or  eggs  with  a  butyric-acid  membrane  into  sea-water  whose 
concentration  is  raised  through  the  addition  of  salt  or  sugar, 
the  diameter  of  the  fertilization  membrane  remains  unaltered 
(while  the  protoplasm  of  the  egg  shrinks).  If  we  dilute  the  sea- 
water  by  adding  distilled  water,  the  diameter  of  the  fertilization 


Fig.  59 


Fig.  60 


Fig.  61 


Fig.  62 


Figs.  59-62. — Collapse  of  the  fertilization  membrane  if  a  liquid  colloid; 
e.g.,  some  liquid  white  of  egg,  is  added  to  the  sea- water.  In  Figs.  59  and  60  very- 
little,  in  Figs.  61  and  62  more,  white  of  egg  was  added  to  the  sea-water.  When 
the  eggs  are  replaced  in  normal  sea-water  the  normal  membrane  is  re-established 
at  onCe. 


membrane  also  remains  unaltered  while  the  protoplasm  of  the 
egg  swells.  This  proves  that  the  fertilization  membrane  is 
permeable  for  water,  sugar,  and  salts,  while  the  protoplasm  of 
the  egg  is  not. 

If  we  add,  however,  any  colloidal  substance,  e.g.,  white  of 
egg  or  blood,  or  even  tannic  acid,  to  the  sea-water,  the  membrane 
collapses  at  once  (Figs.  59-62).  If  the  eggs  are  put  into  sea- 
water  again  which  is  free  from  colloids,  the  membrane  becomes 
spherical  again  almost  at  once.    This  proves  that  the  membrane 


Mechanism  of  Membrane  Formation  209 


is  permeable  to  salts  and  water  but  not  for  colloids.  Hence 
the  addition  of  a  colloid  to  the  external  solution  increases  the 
osmotic  pressure  of  the  latter  (although  only  slightly),  but  this 
difference  is  enough  to  cause  all  the  sea-water  contained  between 
fertilization  membrane  and  protoplasm  to  diffuse  out,  and  as 
a  consequence  the  membrane  collapses  as  shown  in  Figs.  Gl 
and  62.  If  restored  to  normal  sea-water  without  colloids,  the 
membrane  becomes  spherical  again.  These  experiments  suc- 
ceed immediately  after  the  membrane  is  formed. 

3.  These  data  give  us  the  key  to  the  conception  of  the  mech- 
anism of  membrane  formation.     The  fertilization  membrane  is 
perfectly  spherical  in  form.     This  imphes  that  the  membrane 
is  in  the  condition  of  tension.     Since,  then,  the  experiments 
already  described  prove  that  in  Strongylocentrotus  the  fertiliza- 
tion membrane  is  easily  permeable  to  sea-water,  there  must 
prevail  within  the  membrane  an  osmotic   pressure  which  is 
equal  to  the  tension  of  the  membrane.     This  pressure  must  be 
due  to  a  substance  which  is  contained  within  the  membrane 
cavity  and  cannot  diffuse  out  through  it.     This  substance  must 
be  a  colloid.     The  existence  of  such  a  colloidal,  indiffusable 
substance  within  the  membrane  cavity  also  explains  the  above- 
mentioned  fact  that  when  the  membrane  has  been  caused  to 
collapse  by  the  addition  of  serum  to  the  sea-water,  it  can  be 
restored  to  its  normal  condition  of  tension  by  replacing  the  egg 
in  ordinary  sea-water.     Another  fact  that  is  also  explained  by 
this  hypothesis  is  that  the  membrane  does  not  begin  to  collapse 
until  a  certain  definite  mass  of  serum  or  protein  has  been  added 
to  the  sea-water.  1 

We  mentioned  in  the  first  chapter  that  when  the  membrane 
is  slowly  produced  it  can  be  observed  that  this  process  starts 
in  a  roughening  of  the  surface  of  the  egg,  and  that  this  is  due 
to  the  formation  of  numerous  small  drops.  Now  it  appears  to 
me  (so  far  as  the  osmotic  properties  of  the  membrane  are 

1  Loeb,  op.  cit. 


210    Artificial  Parthenogenesis  and  Fertilization 

concerned)  that  this  formation  of  droplets  depends  upon  the  fact 
that  a  colloidal  substance,  which  lies  below  the  surface  layer 
of  the  unfertilized  egg  or  is  secreted  from  the  egg,  suddenly  swells 
by  absorption  of  sea-water.  In  the  typical  case  of  membrane 
formation  this  swelling  results  finally  in  a  complete  liquefaction 
of  the  colloid.  In  other  cases  the  swelling  is  less  complete  and 
the  formation  of  a  gelatinous  film  results. 

4.  If  this  idea  is  correct  it  should  be  possible  to  prove  that 
the  agencies  which  cause  membrane  formation  may  also  cause  a 
swelling  and  liquefaction  of  some  colloidal  substance  associated 
with  the  egg.  This  proof  can  be  furnished  in  the  case  of  the 
chorion  which  surrounds  the  immature  egg  of  Lottia,  a  mollusc 
of  the  Pacific  coast. 

Fig.  63  represents  the  unripe  egg  of  Lottia.  While  in  this 
condition,  the  egg  cannot  be  fertilized  by  a  spermatozoon. 
It  possesses  an  irregular  outline,  owing  to  the  fact  that  it  is 
surrounded  by  a  stout  membrane,  the  so-called  chorion.  When 
this  membrane  is  removed,  the  egg  assumes  the  form  of  a  sphere 
(Fig.  65) .  We  will  now  show  that  the  various  substances  which 
cause  membrane  formation  in  the  sea-urchin  egg  also  cause  a 
swelling  and  liquefaction  of  this  chorion.  Saponin  is,  as  we 
have  seen,  one  of  these  substances.  If  an  unripe  egg  of  Lottia 
is  placed  in  5  c.c.  of  sea-water  to  which  have  been  added  about 
six  drops  of  a  |  of  1  per  cent  solution  of  saponin  (in  sea-water), 
in  some  four  minutes  it  changes  from  the  condition  shown  in 
Fig.  63  to  that  in  Fig.  64,  and  in  about  another  four  minutes 
the  chorion  has  quite  disappeared  and  the  egg  has  become 
spherical  (Fig.  65).  A  comparison  of  Figs.  65  and  64  will  show 
that  under  the  influence  of  saponin  the  chorion  first  swells 
greatly  by  the  imbibition  of  sea-water,  and  finally  liquefies. 

A  second  agency  calling  forth  membrane  formation  in  the 
sea-urchin  egg  is  bases  in  the  presence  of  free  oxygen.  As  long 
as  the  unfertilized  eggs  of  Lottia  lie  in  normal  sea-water  the 
chorion  remains  unaltered.     But  if  the  alkalinity  of  the  sea- 


Mechanism  of  Membrane  Formation 


211 


water  is  raised  by  the  addition  of  some  NaOH  (e.g.,  1.0  c.c. 
N/10  to  50  c.c.  sea-water),  the  chorion  is  gradually  dissolved 
in  a  number  of  eggs.     The  writer  has  recently  found  that  the 


Fig.  63 


Fig.  64 


Fig.  6.5 


Figs.  63-6.5. — Dissolution  of  the  chorion  surrounding  the  egg  of  Lottia 
gigantea,  a  mollusc,  on  treatment  with  saponin.  Fig.  63  shows  the  egg  before  the 
treatment  with  saponin,  c  is  the  chorion.  Fig.  64  shows  the  same  egg  a  few 
minutes  after  the  addition  of  saponin.  The  chorion  c  is  greatly  swollen  and  at 
the  point  of  liquefaction.  Fig.  65  shows  the  chorion  having  conipk-telv  disap- 
peared and  the  egg  become  spherical.  In  this  condition  it  is  permeable  for  the 
spermatozoon. 

weak  bases  are  much  more  efficient  than  the  strong  bases. 
Thus  the  amines  and  NH4OH  will  cause  the  swelling  and  lique- 
faction of  the  chorion  of  Lottia  in  the  same  concentration  much 
more  rapidly  than  the  strong  bases  NaOH  and  tetraethyl- 
ammoniumhydroxide. 


212     Artificial  Parthenogenesis  and  Fertilization 

This  action  of  bases  upon  the  swelHng  and  hquef action  of  the 
membrane  of  Lottia  takes  place  only  in  the  presence  of  oxygen. 
If  the  oxygen  was  removed,  the  eggs  of  Lottia  kept  their 
chorion  and  their  irregular  outline,  even  if  they  remained  for 
from  four  to  six  hours  in  alkaline  sea-water  (50  c.c.  sea-water + 
1.0  c.c.  N/10  NaOH).  If  the  eggs  were  afterward  placed  in 
oxj'genated  alkaline  sea-water  the  chorion  dissolved  and  the 
eggs  could  be  fertilized  by  sperm.  The  addition  of  KCN  to 
the  hyperalkaline  sea-water  also  prevented  the  dissolution  of 
the  chorion.^ 

We  are  forced  to  assume  that  in  our  original  method  of 
artificial  parthenogenesis  the  hypertonic  solution  combined  two 
effects,  the  membrane  formation  and  the  corrective  effect.  The 
membrane  formation  was  atypical,  since  it  only  led  to  the  forma- 
tion of  a  gelatinous  film  around  the  egg,  but  it  was  the  essential 
feature.  It  seems  to  the  writer  to  be  of  great  interest  that  a 
hypertonic  solution  will  also  cause  the  swelling  and  liquefaction 
of  the  chorion  of  Lottia} 

Finally  we  have  seen  that  benzol  causes  membrane  forma- 
tion in  the  sea-urchin  egg;  it  also  causes  the  liquefaction  of  the 
chorion  of  Lottia. 

Acid  did  not  dissolve  the  chorion  of  Lottia,  nor  will  it  cause 
parthenogenesis  in  Lottia  either.  Acids,  however,  dissolve  the 
chorion  of  the  sea-urchin  egg,  as  Herbst  first  observed. 

All  these  facts  led  the  writer  to  speculate  as  to  whether 
the  cortical  layer  of  the  unfertilized  egg  does  not  contain  a 
substance  similar  to  that  of  which  the  chorion  of  these  eggs 
consists;  that  the  membrane  formation  is  only  the  expression 
of  the  swelling  and  liquefaction  of  this  colloidal  substance,  and 
that  the  swelling  and  liquefaction  of  this  substance  is  the 
prerequisite  which  allows  the  egg  to  develop. 

5.  In  my  experiments  I  have  often  had  the  opportunity  of 
observing  membrane  formation  in  eggs  that  were  not  spherical 

1  Loeb,  Untersuchungen  ueber  kunstliche  Parthenogenese,  p.  369,  Leipzig,  1906. 
-  Loeb,  op.  cit. 


Mechanism  of  Membrane  Formation  213 


but  possessed  some  other  shape.     In  such  eggs  the  egg  mem- 
brane at  the  start  followed  the  contour  of  the  egg.     Tliis  proves 
that  the  primary  factor  in  membrane  formation  is  the  swell- 
ing of  a  substance  lying  on  the  surface  of  the  egg,  similar  to 
that  which  forms  the  chorion.     The  swollen  substance  then  be- 
comes more  and  more  liquid.     Immediately  after  the  forma- 
tion of  the  membrane  it  can  be  caused  to  collapse  if  we  add  a 
protein  to  the  sea-water,  which  shows  that  in  S.  purpuratus 
only  liquid  matter  lies  between  membrane  and  protoplasm.     In 
some   cases   of  artificial  parthenogenesis,  e.g.,  by  bases  or  by 
fatty  acids,  in  Arbacia  we  observe  instead  of  a  fertilization  mem- 
brane a  fine  film  surrounding  the  egg.     It  is  possible  that  in 
this  case  the  swelling  is  less  complete  and  the  membrane  re- 
mains gelatinous.     There  may  be  all  kinds  of  transition  stages 
between  the  gelatinous  film  and  the  cases  with  a  typical  fer- 
tilization   membrane    separated    from    the   protoplasm   by    a 
liquid. 

The  existence  of  a  colloid  substance  within  the  membrane 
chamber  can  be  established  by  direct  observation.  For  it  can 
be  seen  that  the  fluid  between  the  membrane  and  cytoplasm 
contains  a  constituent  of  somewhat  higher  refractive  power 
than  the  sea-water. 

6.  We  have  already  mentioned  that  substances  like  ben- 
zol, saponin,  etc.,  can  cause  both  membrane  formation  and 
cytolysis. 

The  first  of  the  two  is  produced  when  they  have  time  to 
affect  only  the  surface  of  the  egg;  cytolysis  is  produced  when 
their  effect  extends  to  the  deeper  layers  of  the  egg. 

Now  the  cases  of  cytolysis  afford  a  ver^'  pretty  demonstra- 
tion of  our  theory.  For  since  the  greater  the  fraction  of  the 
egg  which  comes  under  the  effect  of  the  nuMnbrane-forming 
reagents,  the  greater  the  amount  of  colloid  that  must  be  lique- 
fied; hence  a  greater  osmotic  pressure  should  be  set  up  by  com- 
plete cytolysis  than  by  simple  membrane  formation,  and  hence 


214     Artificial  Parthenogenesis  and  Fertilization 

in  this  case  the  diameter  of  the  egg  should  be  much  greater. 
This  happens  to  be  actually  the  case. 

If  unfertilized  sea-urchin  eggs  are  placed  in  a  weak  saponin 
solution  (in  sea-water),  normal  membrane  formation  takes  place 
after  a  few  minutes;  upon  leaving  the  eggs  longer  in  the  solution, 
however,  cytolysis  ensues,  and  the  diameter  of  the  egg  may 
increase  to  double  its  size.^  The  same  phenomenon  takes  place 
if  fertilized  eggs  are  exposed  to  the  action  of  saponin.  If, 
however,  the  contents  are  coagulated  by  heat  before  the  eggs 
are  exposed  to  the  saponin,  or  a  body  that  has  a  similar  effect, 
this  increase  in  volume  no  longer  occurs.  This  appears  to 
indicate  that  the  colloidal  substance  that  exerts  the  osmotic 
pressure  is  a  protein. 

It  may  be  said  here  that  the  fertilization  membrane  is  insol- 
uVjle  in  benzol,  ether,  alcohol,  saponin,  and  similar  substances. 
Hence  it  is  not  a  lipoid. 

7.  Robertson^  has  extended  these  observations  by  comparing 
the  effect  of  different  proteins  on  the  prevention  of  membrane 
formation  by  butyric  acid.  He  treated  the  eggs  of  S.  purpu- 
ratus  with  butyric  acid,  but  instead  of  putting  them  into  normal 
sea-water  he  brought  them  into  sea-water  to  which  various 
quantities  of  different  soluble  proteins  had  been  added.  His 
results  are  given  in  Table  XXXVIII. 

TABLE  XXXVIII 


Protein 

Highest  Observed  Con- 
centration Which  Per- 
mits the  Formation  of 
a  Spherical  Membrane 

Lowest  Observed  Con- 
centration Wliich  Pre- 
vents the  Formation  of 
a  Spherical  Membrane 

The  mixed  serum  proteins 

Gelatin 

''Insoluble"  serum  globulin..  .  . 
Casein 

3.70 
1.00 
0.30 
0.25 
0.125 

7.40 
2.00 
0.60 
0  50 

Ovomucoid 

0.25 

1  See  Figs.  39  and  45,  chap.  xvii. 

2  Robertson,   Archiv  f.  Entwicklungsmechanik,  XXXV,  80,   1912. 


Mechanism  of  Membrane  ForxMation  215 


Robertson  states  that  this  order  of  inhil)iting  efficiency  o( 
various  proteins  is  the  reverse  of  their  abihty  to  pass  through 
a  porcelain  filter. 

In  striking  confirmation  of  the  view  expressed  by  Loel;  that  the 
formation  of  the  membrane  in  fertilized  eggs  is  due  to  the  osmotic 
imbibition  of  water  by  the  egg,  and  that  the  inhil)iting  effect  of  colloids 
upon  their  formation  is  due  to  their  inability  to  pass  through  the  mem- 
brane, we  find  that  the  order  of  effectiveness  of  the  various  proteins 
in  inhibiting  membrane  formation  is  the  reverse  order  of  their  ability 
to  pass  through  a  porcelain  filter. 

8.  Membrane  formation  by  acids  or  saponin  or  benzol  is 
not  dependent  upon  the  presence  of  free  oxygen.  As  I  showed 
seven  years  ago,  an  egg  is  not  deterred  by  KCN  from  forming!; 
a  membrane  under  the  influence  of  acid.  I  left  unfertilized 
sea-urchin  eggs  at  15°  C.  for  several  hours  (up  to  24  hours)  in  a 
mixture  of  50  c.c.  of  sea-water +2  c.c.  1/20  per  cent  KCN  and 
added  sperm  thereto.  The  eggs  at  once  formed  perfect  fertili- 
zation membranes  in  the  cyanide  sea-water.^  But  the  develoj)- 
ment  of  the  eggs  was  completely  inhibited  by  such  a  solution. 
The  development  of  the  eggs  requires  free  oxygen,  but  mem- 
brane formation  by  fatty  acid,  on  the  other  hand,  does  not. 

In  the  case  of  artificial  parthenogenesis  by  bases,  however, 
oxidation  is  required  for  membrane  formation.  It  is  possible 
that  in  this  case  the  process  of  oxidation  leads  to  the  formation 
of  a  substance  w^hich  causes  the  physical  change  underlying 
membrane  formation. 

9.  What  has  the  membrane  formation  to  do  witli  the 
development?  The  writer  published  in  1905  the  following 
hypothesis : 

From  all  these  facts  mentioned  here,  I  liave  gathereil  the  impres- 
sion that  the  membrane  formation,  or  possibly  the  process  whieii  results 
in  membrane  formation,  is  an  essential  feature  of  the  process  of  fertiliza- 
tion not  only  in  the  sea-urchin  egg  but  also  in  at  k^ist  eertain  starfish 

1  Loeb,  "Der  chemische  Charakter  des  Befruclituii^svorganK*"^,"    Hicul.em 
Zeitschr.,  I,  191.  1906. 


216     Artificial  Parthenogenesis  and  Fertilization 


e.g.,  Asteritia.  It  maj^  seem  pedantic  to  discriminate  between  the 
membrane  formation  and  the  process  underlying  it:  but  this  discrimi- 
nation  is  suggested  by  a  suspicion  on  my  part  that  the  membrane 
formation  is  the  result  of  a  process  of  secretion  of  a  liquid  from  the 
egg;  and  that  this  secretion  or  the  throwing-out  of  certain  substances 
of  the  egg  is  the  important  feature,  while  the  lifting-up  of  the  surface 
layer  of  the  egg  (the  membrane  formation  proper)  is  only  a  mechanical 
consequence  of  this  secretion,  but  of  no  importance  in  itself  .^ 

The  same  idea  was  repeated  by  the  writer  a  little  later  in 
the  following  words:  "We  might  think  of  the  possibility  that 
an  elimination  of  a  definite  inhibiting  substance  sets  into  motion 
the  chemism,  which  underlies  development."-  On  this  assump- 
tion the  colloidal  substance  which  undergoes  the  swelling  would 
be  the  substance  whose  removal  gives  rise  to  the  development. 

F.  Lillie^  has  recently  found  that  a  layer  of  substance, 
which  in  the  unfertilized  egg  of  Nereis  lies  under  the  natural 
vitelline  membrane  of  the  egg,  flows  out  (is  ''secreted")  and 
forms  a  thick  gelatinous  layer  around  the  egg,  as  soon  as  the 
spermatozoon  comes  in  contact  with  the  egg.  But  this  gelati- 
nous layer  resembles  the  gelatinous  envelope  which  surrounds 
the  frog  egg  and  does  not  form  a  tough  membrane  at  its  outer 
surface,  such  as  we  observe  in  the  sea-urchin  egg. 

Lillie  assumes 
that  the  presence  of  this  colloid  substance  in  the  cortex  is  an  inhibition 
to  the  maturation  of  the  egg,  because  as  soon  as  it  is  removed,  matura- 
tion processes  are  set  in  motion  and  both  polar  bodies  formed.  In 
what  manner  it  inhibits  is  of  course  problematical.  In  the  egg  of 
Ascaris  megalocephala  there  is  a  similar  excretion  of  a  cortical  colloid 
which  forms,  in  this  case,  the  thick  resistant  perivitelhne  membrane. 
The  appearance  of  the  fertilization  membrane  of  echinids  might  be 
similarly  due  to  excretion  of  a  cortical  colloid  which  is  removed  by 
diffusion  and  hence  is  not  detected. 

1  Loeb,  "Artificial  ISIembrane  Formation  and  Chemical  Fertilization  in  a 
Starfish,"  University  of  California  P ublications,  Physiology ,  II.  154, 1905;  reprinted 
in  U liter suchung en  zur  kilnstlichen  Parthenogenese,  p.  362,  Leipzig,  1906. 

2  Loeb,  "Die  kunstliche  Parthenogenese,"  Oppenheimer's  Handbuch  der  Bio- 
chemie,  II,  100,  1909. 

i  Lillie,  Jour.  Morphol.,  XXII,  361,  1911. 


Mechanism  of  Membrane  Formation  217 

Bataillon  has  expressed  a  similar  view,  but  both  authors 
have  apparently  overlooked  the  writer's  former  statements. 
I  have  recently  omitted  to  emphasize  this  idea  of  a  secretion 
since  it  is  merely  hypothetical  and  moreover  the  word  ''secre- 
tion" is  not  clear  from  a  phj^sicochemical  viewpoint.  I 
therefore  preferred  the  expression  ''cytolysis  of  the  cortical 
layer  of  the  egg, "  which  is  a  clearer  expression  of  what  actually 
occurs  and  which  is  better  understood  from  a  physicochemical 
viewpoint.  It  may  be  that  the  action  of  cytolytic  agents  upon 
the  egg  merely  induces  secretion,  or  that  secretion  is  in  all  cases 
in  the  ultimate  analysis  a  form  of  cytolysis. 

Since,  however,  the  rise  of  the  rate  of  oxidations  is  the  essen- 
tial feature  in  the  causation  of  development  in  the  egg  of  the 
sea-urchin;  and  since  this  rise  is  also  brought  about  by  com- 
plete cytolysis  of  the  egg,  it  seems  safer  to  say  that  the 
cytolysis  of  the  cortical  layer  of  the  egg  (which  results  in 
membrane  formation)  is  the  essential  feature  in  the  causation 
of  development. 

10.  Harvey  has  expressed  the  view  that  the  essential  con- 
dition for  the  formation  of  the  membrane  is  an  increased  per- 
meability of  the  egg  surface  for  a  membrane  substance  which 
passes  out  and  hardens  to  a  membrane  in  contact  with   sea- 
water.^     Ries2  and  Elder^  assume  that  this  hardening  occurs 
in  contact  with  the  chorion  which  surrounds  the  egg.     It  is 
possible  that  the  droplets,  which  initiate  the  membrane  forma- 
tion quite  frequently  in  the  egg  of  S.  purpiiratus,  are  really-  a 
secretion  of  a  colloid  which  upon  coming  in  contact  with  sea- 
water   swells  by  absorbing  sea-water  and  whicli   iiardens  at 
its  outer  surface  to  the  characteristic  membrane.     Still,  one 
wonders  why  this  should  be  called  a  secretion,  since  comi)lete 
cytolysis  of  the  egg    by    saponin    also    results    in   membrane 
formation. 

1  Harvey,  Jour.  Exper.  Zool.,  VIII,  .355.  1910. 
2Ries,  Centralbl.  f.  Physiol..  XXIII,  369,  1909. 
3  Elder,  Archiv /.  Entwicklunysmechanik,  XXXV,  145,  1912. 


218     Artificial  Parthenogenesis  and  Fertilization 


Another  hypothetical  suggestion  is  that  the  fertilization 
membrane  is  preformed  in  the  unfertilized  egg  and  is  merely 
the  peripheral  film  of  protoplasm  which  is  lifted  up  from 
the  egg  through  the  swelling  and  liquefaction  of  some  protein 
lying  underneath  in  the  cortical  layer  of  the  egg.  When  lifted 
up  from  the  egg  the  preformed  membrane  undergoes  a  modifica- 
tion; it  becomes  thicker  and  tougher.  The  objection  has  been 
raised  that  no  such  surface  film  is  visible  in  the  unfertilized  egg, 
but  this  objection  is  not  valid,  since  the  surface  films  which 
form  on  the  principle  of  Thomson-Gibbs  are  beneath  the  limit 
of  visibility.  This  fact  also  meets  the  objection  of  Moore,^ 
that  after  shaking  unfertilized  eggs  into  fragments,  each  frag- 
ment can  still  form  a  fertilization  membrane  upon  the  entrance 
of  a  spermatozoon,  since  each  fragment  is  bound  to  have  at  its 
surface  such  a  film.  But  we  are  now  in  the  realm  of  mere 
hypotheses  to  which  it  is  not  worth  while  to  devote  much  space. 

1  Moore,  University  of  California  Publications,  Physiology,  IV,  89,  1912. 


XXI 

IS  DEVELOPMENT  OF  THE  SEA-URCHIN  EGG  POSSIBLE 

WITHOUT  MEMBRANE  FORMATION  OR  WITHOUT 

THE  SECOND  (CORRECTIVE)  FACTOR? 

1.  It  has  been  known  for  a  long  time  that  if  sea-urchin  eggs 
lie  for  some  time  in  sea-water  they  may  begin  to  segment.  I 
have  recently  investigated  this  phenomenon  in  the  eggs  of 
S.  purpuratus,  and  find  that  such  a  segmentation  is  always 
preceded  by  membrane  formation.^ 

If  the  unfertilized  eggs  of  S.  purpuratus  are  kept  at  a  rela- 
tively low  temperature,  one  notices  that  after  twenty-four  to 
forty-eight  hours,  in  a  part  of  the  eggs  of  certain  females,  a 
membrane  formation  appears  of  that  type  which  is  common  in 
the  eggs  of  Arbacia  after  an  acid  treatment.     Around  the  eggs 
a  gelatinous  layer  is  formed.     If  such  eggs  are  kept  at  a  low 
temperature  and  with  sufficient  oxygen  supply,  they  begin  to 
segment  and  this  segmentation  may  proceed  to  the  eight-  or 
sixteen-cell  stage  almost  regularly  (Figs.  66-72).     Only  those 
eggs  segment  that  form  a  membrane.     The  eggs  of  certain 
females  only  show  this  tendency  and  the  number  of  purpuratus 
females  whose  eggs  form  membranes  spontaneously  is  very 
small.     It  can  be  shown  that  the  eggs  which  form  membranes 
spontaneously  behave  in  the  same  way  as  the  eggs  in  which 
the  membrane  formation  is  called  forth  by  butyric-acid  treat- 
ment.    At  a  high  temperature  they  disintegrate  at  the  time  of 
the  first  segmentation;  at  a  low  temperature  they  can  segment. 
Moreover,  the  eggs  which  form  membranes  spontaneously  can 
be  caused  to  develop  into  larvae  by  a  short  treatment  with 
hypertonic  sea-water. 

The  question  arises,  What  causes  this  spontaneous  membrane 

»  Loeb,  Archiv  f.  EntwicklunQsmechanik,  XXXVI.  G26.  1913. 

219 


220     Artificial  Parthenogenesis  and  Fertilization 


formation  if  eggs  lie  for  some  time  in  sea-water  ?  No  definite 
answer  can  be  given.  The  effect  may  possibly  be  due  to  the 
action  of  the  NaOH  contained  in  the  sea-water. 

2.  In  his  experiments  on  inducing  development  by  foreign 
blood,  the  writer  made  the  following  observation.  The  unferti- 
lized eggs  of  a  sea-urchin  were  placed  in  a  mixture  of  1  c.c. 


Figs.  66-72. — Camera  drawings  of  unfertilized  eggs  of  S.  ptirpuratus  which 
had  begun  to  segment  spontaneously.  In  all  the  eggs  a  fine  gelatinous  membrane 
was  formed. 

of  pig's  serum  and  1  c.c.  of  SrCl2  isotonic  with  sea-water.  After 
five  minutes  this  solution  was  pipetted  off  and  replaced  with 
sea-water,  which  process  was  repeated  four  times.  The  eggs 
were  left  in  the  watch  glasses  for  four  hours  and  then  trans- 
ferred to  a  larger  bowl  of  sea-water.  Only  a  small  number  of 
the  eggs  had  formed  membranes,  and  these  perished  after  a 
little  while  in  the  early  segmentation  stages.  But  a  few  of  the 
other  eggs,  which  had  apparently  not  formed  membranes,  began 
to  divide  and  they  developed  into  normal  swimming  larvae, 
perishing  in  the  blastula  stage.     Similar  results  were  obtained 


Development  without  Membrane  Formation       221 

also  with  a  mixture  of  sea-water  and  pig's  serum  without 
the  addition  of  SrCl^.'  But  at  this  time  the  writer  was  not 
yet  famiHar  with  the  gelatinous  form  of  membranes  in  the 
eggs  of  this  species,  and  so  he  must  leave  it  in  doubt  whether 
or  not  gelatinous  membranes  were  formed  in  the  eggs  which 
developed. 

The  following  hitherto  unpublished  experiments  are  rather 
curious.  Unfertilized  eggs  (without  membranes)  were  placed 
in  m/2  sodium  but^Tate  solution  and  taken  out  from  it  at  differ- 
ent intervals.  The  solution  was  strongly  alkaline  (requiring 
4.2  c.c.  N/10  HCl  [per  50  c.c.  of  solution]  to  turn  neutral  red 
from  3'ellow  to  red). 

A  large  number  of  the  eggs  formed  membranes,  but  most  of 
these  (if  not  all)  succumbed  to  cytolj'sis.  However,  a  small 
number  (about  1  per  cent)  of  the  eggs  removed  after  between 
three  and  four  hours  developed  into  swimming  larvae.  These 
eggs  possessed  either  no  membrane  or  only  one  that  adhered 
very  closely  to  the  egg,  probably  a  gelatinous  membrane.  The 
experiment  was  repeated  with  a  sodium  but}Tate  solution  of 
much  diminished  alkalinity.  The  amount  of  cytolysis  (and 
membrane  formation)  was  reduced  by  this,  but  the  activation 
of  a  few  eggs  took  place  in  this  case  also  (but  not,  of  course, 
before  the  eggs  had  been  transferred  to  normal  sea-water). 
About  2  per  cent  of  the  eggs  that  had  been  about  six  or  seven 
hours  in  the  sodium  butyrate  solution  developed  into  swinuning 
larvae.  1  believe  that  in  both  these  cases  a  gelatinous  mem- 
brane was  formed,  but  the  remarkable  fact  is  that  the  eggs 
developed  to  the  blastula  stage  at  room  temperature  without 
subsequent  treatment  with  a  hypertonic  solution.  Could  it  be 
possible  that  the  butyrate  solution  acted  like  a  solution  in 
which  oxidations  were  prevented  ? 

3.  E.   P.   Lyon  succeeded   in   causing  artificial   partheno- 
genesis in  Arbacia  pustulata  and  Strongyloccntrotus  lividus  at 

1  Loeb,    Pfluger's    Archiv,    CXXIV,    50,    1«)0S. 


222     Artificial  Parthenogenesis  and  Fertilization 

Naples  by  treating  their  eggs  with  hypertonic  sea-water.^ 
He  also  investigated  the  effect  of  acids. 

Hydrochloric  acid  has  been  found  by  Loeb  to  be  an  efficient 
reagent  for  causing  artificial  parthenogenesis  in  starfish.  He  found  it 
did  not  succeed  in  Arbacia  punctulata  (at  Woods  Hole).  But  strangely 
enough  it  is  one  of  the  best  reagents  I  found  for  Arbacia  pustulata 
(at  Naples).  Usually  2,  3,  4,  5,  6,  and  7  c.c.  of  a  N/10  solution  of 
hydrochloric  acid  in  sea-water  was  added  respectively  to  dishes 
containing  100  c.c.  of  sea-water.  Eggs  immersed  in  these  solutions 
were  taken  out  at  intervals  of  from  two  to  fifteen  minutes.  Some  of  the 
best  results  were  obtained  from  2  c.c.  acid  in  100  c.c.  sea-water,  ten  to 
fifteen  minutes'  exposure;  3  c.c.  acid,  seven  to  twelve  minutes' 
exposure;  4  c.c,  acid,  nine  minutes'  exposure;  7  c.c.  acid,  five  minutes' 

exposure In  the  best  experiments  perhaps  10  per  cent  of  the 

eggs  developed  into  swimming  larvae.  Manj^  of  these  swam  up  to 
the  top  of  the  liquid,  just  like  the  larvae  from  fertihzed  eggs.  They 
formed  fully  developed  plutei  which  Uved  as  long  as  individuals  pro- 
duced from  fertihzed  eggs  and  kept  under  the  same  conditions. 

No  positive  results  were,  however,  obtained  by  this  method 
at  Naples  with  S.  lividus,  but  Lyon  succeeded  in  obtaining  a 
couple  of  larvae  b}'  treating  the  unfertilized  eggs  of  S.  lividus 
with  carbonic  acid  in  sea-water.  The  importance  of  membrane 
formation  from  the  point  of  view  of  development  was  not  recog- 
nized at  that  time,  but  I  believe  that  the  eggs  in  Lyon's  experi- 
ment formed  a  gelatinous  membrane. 

We  may  as  well  point  out  here  that  the  eggs  of  S.  purpuratus 
and  frandscanus  at  Pacific  Grove  cannot  be  made  to  develop 
into  larvae  by  a  mere  treatment  wdth  acid  unless  they  are  kept 
at  a  very  low  temperature.  In  this  respect,  there  is  a  quali- 
tative difference  between  the  eggs  of  the  European  and  Cali- 
fornian  sea-urchins. 

This  difference  between  the  behavior  of  the  eggs  of 
Strongylocentrotus  at  Naples  and  in  California  is  also  corrobo- 

lE.  P.  Lyon,  "Experiments  in  Artificial  Parthenogenesis,"  Am.  Jour. 
Physiol.,  IX,  308,   1903. 


Development  without  Membrane  Formation      223 

rated  by  some  work  of  Herbst^  to  which  we  shall  return  later. 
Herbst  put  the  eggs  of  Sphaerechinus  in  50  c.c.  of  sea-water +3c.c. 
N/10  acetic  acid  for  two  to  eight  minutes.  The  eggs  formed, 
if  I  interpret  Herbst  correctly,  not  a  typical  membrane,  but  a 
fine  gelatinous  film,  and  upon  transference  to  normal  sea-water 
a  few  of  them  developed  into  larvae,  without  it  being  necessary 
to  expose  them  first  to  hypertonic  sea-water.  I  am  inclined  to 
believe  that  in  all  cases  in  which  an  unfertilized  sea-urchin  egg 
has  been  caused  to  develop,  a  typical  or  atypical  membrane  had 
been  formed. 

The  reader  will  notice  that  in  these  cases  the  eggs  developed 
without  any  treatment  with  a  hypertonic  solution,  at  room 
temperature.  We  shall  see  later  that  this  is  not  uncommon 
in  the  egg  of  the  starfish.  We  must  conclude  that  in  such  cases 
the  corrective  effect  is  produced  by  changes  taking  place  inside 
the  egg  itself.  The  situation  is  comparable  to  that  in  the 
experiments  in  which  the  hypertonic  solution  was  replaced  by 
a  treatment  of  the  eggs  with  lack  of  oxygen.  In  this  case  we 
are  also  forced  to  assume  that  the  egg  itself  was  able  to 
produce  the  substance  which  counteracts  the  threatening  disin- 
tegration. In  the  eggs  of  some  species,  and  possibly  of  some 
strains  or  individuals,  this  substance  can  possibly  be  formed 
under  normal  conditions  and  for  such  eggs  the  process  of  mem- 
brane formation  may  suffice,  the  egg  being  able  to  furnish  the 
corrective  effect  or  being  naturally  more  resistant. 

1  Herbst,  "  Vererbungstudien  IV,"  Archiv  f.  Entwicklungsmechanik,  XXII, 
473,  1906. 


XXII 

THE  ACTION  OF  THE  SPERMATOZOON  UPON  THE  EGG 

I.       HETEROGENEOUS    HYBRIDIZATION 

1.  In  1905  the  writer  showed  that  in  the  sea-urchin  ogg 
artificial  parthenogenesis  is  produced  by  two  agencies,  one  of 
which  causes  membrane  formation  while  the  second  one  serves 
to  make  the  development  more  normal.  In  1906  he  expressed 
the  idea  that  the  spermatozoon  also  caused  the  development 
by  two  different  agencies,  one  of  which  induced  membrane 
formation  while  the  other  one  acted  as  a  corrective — like  the 
hypertonic  solution.^  We  will  now  consider  the  proof  for  this 
statement. 

The  eggs  of  the  sea-urchin  cannot  be  fertilized  by  the  sperm 
of  the  starfish  under  normal  conditions.  In  1903  the  writer 
found  that  the  eggs  of  S.  purpuratus  can  easily  be  fertilized  by 
the  sperm  of  the  starfish  if  we  render  the  sea-water  a  little 
more  alkaline.  If  0.6  c.c.  N/10  NaOH  is  added  to  50  c.c. 
sea-water  and  sperm  of  a  starfish  (Asterias)  is  added,  all  the 
eggs  form  a  typical  fertilization  membrane.^ 

After  adding  the  sperm  of  Asterias  to  the  eggs  of  S.  purpur- 
atus, it  is  noticeable  that  not  all  the  eggs  which  form  membranes 
develop  into  larvae.  Some  of  them  begin  to  segment  after  the 
usual  interval,  but  others  behave  like  eggs  which  have  only 
formed  membranes  under  the  influence  of  butyric  acid.  For  at 
15°  C.  they  begin  to  proceed  toward  nuclear  division,  but  then 
most  of  them  go  to  pieces  within  a  few  hours.  I  thought  at 
first  that  in  these  eggs  we  were  dealing,  not  with  an  influence  of 
the  spermatozoon,  but  with  the  effect  of  the  extraneous  body 

1  Loeb,  "Versuche  ueber  den  chemischen  Charakter  des  Befruchtungsvor- 
ganges,"  Biochem.  Zeitschr.,  I,  183,  1906.  y 

=  Loeb,  "Weitere  Versuche  ueber  hetero^ene  Hybridisation  bei  Ecliino- 
dermen,"  PflUger's  Archiv,  CIV,  325,  1904. 

225 


226     Artificial  Parthenogenesis  and  Fertilization 


fluids  mixed  with  the  sperm.     Control  experiments,  however, 
showed  that  this  was  not  the  case  with  the  eggs  of  S.  purpuratus. 

In  one  experiment,  all  the  eggs  of  a  female  purpuratus  formed 
membranes  when  placed  in  a  mixture  of  50  c.c.  of  sea- water + 
0.6  c.c.  N/10  NaOH,  to  which  was  added  only  a  few  drops 
of  living  Asterias  sperm.  However,  only  a  few  of  these  eggs 
(only  a  fraction  of  1  per  cent)  developed  to  larvae;  the  others 
disintegrated  in  the  manner  characteristic  of  eggs  which  have 
not  been  treated  with  a  hypertonic  solution  subsequently  to 
artificial  membrane  formation.  This  membrane  formation, 
however,  was  brought  about  in  this  case  through  the  living 
spermatozoa  and  not  by  the  body  fluids  of  the  starfish;  this 
point,  which  is  important,  was  established  by  adding  to  the 
same  eggs  sperm  from  the  same  Asterias  male  after  heating  it 
to  50°,  in  which  case  no  egg  formed  a  membrane.  Even  on 
addition  of  ten  times  the  amount  of  (dead)  sperm  which  was 
sufficient  in  the  living  condition  to  cause  all  the  eggs  to  form 
membranes,  membranes  were  formed  by  only  a  few  of  the 
eggs,  even  after  a  duration  of  several  hours  and  when  the  eggs 
were  thoroughly  shaken  and  mixed  with  the  sperm.  Hence 
the  membranes  in  the  above-mentioned  experiments  were 
formed  by  the  living  spermatozoa  and  not  by  any  admixture 
with  the  sperm. 

While  all  the  eggs  of  S.  purpuratus  form  membranes  under 
the  influence  of  living  sperm  within  an  hour,  only  a  fraction  of 
the  eggs  (which  varies  in  different  cases)  develops  into  larvae. 
The  rest  of  the  eggs  behave  as  if  the  sperm  had  only  brought 
about  the  artificial  membrane  formation  by  the  giving-off  to 
the  eggs  of  a  substance  which  acts  like  butyric  acid.  The 
explanation  of  this  result  is  probably  as  follows.  The  sperm  of 
the  starfish  penetrates  only  slowly  into  the  sea-urchin  egg;  the 
starfish  spermatozoon  lingers  for  a  long  time  in  contact  with 
the  protoplasm  of  the  sea-urchin  egg  before  it  gets  into  the  in- 
terior.    The  time  during  which  the   spermatozoon  lingers  in 


Action  of  the  Spermatozoon  upon  the  Egg       227 


contact  with  the  surface  of  the  protoplasm  before  entering 
varies  in  different  cases.  If  the  time  is  long  enough  the  mem- 
brane-forming substance  may  be  given  off  to  the  egg  and  the 
membrane  formed,  before  the  spermatozoon  has  entered  com- 
pletely. The  formation  of  the  membrane  throws  the  sperma- 
tozoon out  of  the  egg  and  prevents  its  entrance  permanently, 
since  no  spermatozoon  can  penetrate  the  fertilization  mem- 
brane. Such  eggs  behave  as  if  they  had  only  undergone  mem- 
brane formation  by  butyric  acid.  They  begin  to  develop  but 
soon  perish.  If,  however,  the  spermatozoon  penetrates  through 
the  surface  of  the  protoplasm  before  the  membrane-forming 
substance  has  had  time  to  act  the  egg  can  develop.  In  this 
case,  in  addition  to  the  membrane-forming  substance,  the 
corrective  substance  has  penetrated  into  the  egg. 

This  idea  was  supported  by  the  following  observation. 

Eggs  of  a  purpuratus  were  treated  with  living  Asterias  sperm  in 

hjTDeralkaline  sea-water,  and  all  formed  membranes.     Some  of 

the  eggs  were  left  in  normal  sea-water  as  a  control ;  the  majority, 

however,  were  placed  after  an  hour  in  hypertonic  sea-water 

(50  c.c.  sea-water -F8  c.c.  2|  m  NaCl)  and  at  different  intervals 

transferred  to  normal  sea-water.     Of  the  control  eggs,  about  a 

third  developed  into  larvae,  while  the  remaining  two-thirds 

behaved  like  eggs  treated  only  with  butyric  acid;   they  began 

to  develop  and  then  disintegrated.     On  the  other  hand,  the 

eggs  which  had  been  exposed  for  about  30  to  40  minutes  to 

hjrpertonic  sea-water  all  developed  into  normal  larvae.     Dead 

Asterias  sperm   was   absolutely   ineffective.     This   proves,   it 

seems  to  me,  in  the  most  striking  manner  that  the  spermatozoon 

also  induces  the  development  of  the  egg  through  two  agencies, 

viz.,  a  membrane-forming  substance,  a  "lysin"  and  a  second 

agent,  which  has  the  same  effect  as  the  hypertonic  salt  solution 

in  our  method  of  artificial  parthenogenesis.     It  is  only  when 

both  factors  contained  in  the  spermatozoon  get  into  the  egg 

that  the  sea-urchin  egg  develops  into  a  larva.     I  am  unable  to 


228    Artificial  Parthenogenesis  and  Fertilization 

suggest  what  may  be  the  nature  of  the  second  factor  in  the 
spermatozoon.  The  ''lysin"  of  the  spermatozoon  serves  for 
the  production  of  membrane  formation.  The  second  factor 
serves  to  turn  the  development  into  the  right  direction  by  the 
suppression  of  the  tendency  to  disintegrate.^ 

2.  A  second  proof  for  the  fact  that  the  spermatozoon  causes 
the  development  of  the  egg  by  two  agencies,  one  of  which  causes 
merely  membrane  formation,  is  contained  in  the  following  facts. 
The  eggs  of  S.  frandscanus  can  be  more  easily  caused  to  form 
membranes  than  the  eggs  of  S.  purpiiratus.  I  found  in  1908  and 
1909  that  if  we  add  living  spermatozoa  of  the  shark  or  of  fowl 
to  such  eggs,  the  eggs  form  membranes.^  In  the  case  of  the 
spermatozoa  of  the  shark  it  was  possible  to  wash  them  first 
repeatedly  in  sea-water  and  thus  free  them  from  all  blood  or 
lymph.  Nevertheless,  the  eggs  of  S.  frandscanus  formed  ferti- 
lization membranes  upon  contact  with  the  living  spermatozoa 
of  the  shark.  Such  eggs,  when  left  to  themselves,  began  to 
segment  but  very  soon  disintegrated.  If  they  were,  however, 
treated  afterward  with  a  hypertonic  solution  they  developed 
into  larvae.  The  explanation  of  this  fact  is  that  the  living 
heterogeneous  spermatozoon  upon  contact  with  the  egg  gives 
off  to  the  latter  the  membrane-forming  substance,  without 
supplying  the  corrective  effect. 

A  third  proof  lies  in  the  fact,  mentioned  in  a  previous  chap- 
ter, that  the  watery  extract  of  foreign  sperm  calls  forth  the  mem- 
brane formation  in  the  same  way  as  butyric  acid  does  without 
supplying  the  second  corrective  factor. 

3.  I  have  tried  in  vain  to  separate  in  the  same  way  the 
membrane-forming  substance  from  the  living  sperm  of  the  sea- 

1  In  order  to  test  this  idea  further  I  asked  Dr.  Elder  to  make  a  cytological 
examination  of  these  eggs.  He  found  that  when  only  a  few  eggs  of  S.  purpuratus 
which  had  formed  membranes  developed  into  larvae  a  small  percentage  showed 
the  sperm  nucleus;  while  the  otlier  eggs  had  no  sperm  nucleus  although  they  had 
formed  a  membrane. 

-  Loeb,  Address  at  the  International  Congress  of  Medicine,  Budapest,  1909; 
reprinted  in  The   Mechanistic  Conception  of  Life,  1912. 


Action  of  the  Spermatozoon  upon  the  Egg       229 

urchin.  I  believe  the  reason  to  be  this,  that  as  soon  as  the 
spermatozoon  of  S.  purptiratus  touches  the  protophism  (the 
fertihzation  cone)  of  the  egg  of  the  same  species  it  is  taken  so 
quickly  into  the  interior  of  the  egg  that  it  is  already  safely 
mside  by  the  time  the  membrane-forming  substance  can  act. 
If  we  fertilize  the  eggs  of  S.  purpuratus  with  their  own  sperm, 
in  about  two  minutes  or  less  all  the  eggs  have  formed  a  ferti- 
lization membrane.  If  we  fertilize  them  with  starfish  sperm. 
It  takes  from  ten  to  sixty  minutes  to  bring  about  the  same 
result.  I  am  inclined  to  see  in  this  difference  of  time  the 
reason  why  it  is  possible  to  separate  the  two  agencies  in  the 
living  sperm  of  the  starfish,  but  not  in  that  of  the  sea-urchin. 

4.  Very  striking  experiments  have  recently  been  carried 
out  by  Oskar  Hertwig  with  Gunther  and  Paula  Hcrtwig,  on 
the  effects  of  radium  on  sperm.  When  the  spermatozol  of 
the  frog  were  exposed  a  short  time  to  radium  before  they  were 
added  to  the  eggs,  the  eggs  were  sickly  and  died  in  the  early 
stages  of  development.  When,  however,  the  spermatozoa  were 
exposed  a  longer  time  to  the  radium,  the  eggs  could  develop 
much  better  and  the  larvae  were  able  to  live  as  long  as  two 
weeks.  This  paradox  finds  its  explanation  in  the  fact  that  when 
the  spermatozoa  had  been  exposed  a  longer  time  to  the  radium 
they  were  able  to  enter  the  egg,  but  the  sperm  nucleus  was  no 
longer  able  to  fuse  with  the  egg  nucleus.  The  spermatozoon 
thus  imparted  only  the  developmental  influences  to  the  eggs,  but 
not  the  hereditary  effects.  In  order  to  produce  this  result  it  was 
necessary  to  expose  the  spermatozoa  for  four  hours  to  60  mg. 
radium  bromide  or  for  twelve  hours  to  10  mg.  radium  bromide.^ 

TvvvT^'^Q^f'^'Af^?''^^^'^  tierischer  KeimzeUen,"  Arch.  f.  mikr.  Anat., 
LXXMI.  1911  Abteilung  f.  Zeugungslehre ;  "Die  Radiumstrahlung  in  ihrer 
Wirkung   auf  die   Entwieklung   tierischer  Eier."    Sitzgsber.    Akad.    Berlin,    1910- 

Mesothoriumversuche  an  tierischen  Keimzellen,  ein  experimenteller  Beweis 
fur  die   Idioplasmanatur  der   Kernsubstanzen/'     Sitzgsber.    Akad.    Berlin,    1911- 

Radiumbestrahlung  unbefruchteter  Froscheier  und  ihre  Entwieklung  nach 
Befruchtung  mit  normalem  Samen."  Arch.  f.  mikr.  Anat.,  LXXVII  1911  "Ver- 
anderung  der  idioplasmatischen  Beschaffenlieit  der  Samenfaden  durch  physikal- 
ische  und  durcli  chemische  Eingriffe."  Sitzgsber.  Akad.  Berlin    191'^ 


230     Abtificial  Parthenogenesis  and  Fertilization 

5.  F.  Lillie^  has  made  some  interesting  experiments  on  the 
fertilization  of  the  egg  of  Nereis,  an  annehd.  It  seems  that 
in  this  egg  the  spermatozoon  comes  in  contact  with  the  fertiliza- 
tion cone  of  the  egg  and  lingers  here  for  more  than  half  an 
hour  before  it  enters.  The  first  contact  of  the  spermatozoon 
with  the  fertilization  cone  leads  to  the  extrusion  of  a  gelatinous 
mass  which  in  the  unfertilized  egg  lies  under  the  perivitelline 
membrane.  Lillie  showed  that  by  centrifuging  the  eggs  at 
this  stage  the  spermatozoon  can  be  thrown  off.  In  this  case  no 
further  development  of  the  egg  follows.  It  should  be  pointed 
out  that  this  contact  action  of  the  spermatozoon  in  Nereis  is 
not  comparable  to  the  results  of  artificial  membrane  formation 
in  the  egg  of  the  sea-urchin,  since  in  the  latter  case  the  egg  not 
only  begins  to  segment  but  may  reach  the  blastula  stage  if  the 
temperature  is  low. 

6.  Kupelwieser^  succeeded  in  causing  the  development  of 
the  egg  of  sea-urchins  by  the  sperm  of  molluscs,  Mytilus, 
Madra,  and  Patella,  and  of  annelids,  Auduinia.  In  this  case 
the  spermatozoa  entered  the  egg  and  often  more  than  one 
spermatozoon  entered,  but  no  fusion  of  sperm  and  egg  nucleus 
occurred.  As  a  rule  the  first  segmentation  began  only  many 
hours  (4  to  20)  after  the  addition  of  sperm.  The  development 
was  most  abnormal  and  no  plutei  were  obtained,  as  far  as  the 
writer  is  able  to  judge.  Kupelwieser  states  that  in  these  experi- 
ments no  fertilization  membrane  is  formed.  If  a  fertilization 
membrane  formed,  the  eggs  did  not  develop. 

The  writer  found  in  1908  that  the  eggs  of  the  sea-urchin 
S.  franciscanus  could  be  fertilized  by  the  sperm  of  a  mollusc, 
Chlorostoina,  and  that  normal  plutei  oi  franciscanus  were  formed. 
His  attempts  in  later  years  to  obtain  plutei  failed. 

IF.  Lillie,  "Studies  of  Fertilization  in  Nereis,"  Jour.  Morphol.,  XXII,  361, 
1911. 

2  Kupelwieser,  Biol.  Centralbl.,  XXVI,  744,  1906;  Archiv  /.  Entwicklungs- 
mechanik,  XXVII,  434.  1909;  Sitzungsber.  d.  Gesellsch.  f.  Morphol.  u.  Physiol, 
in  Miinchen,   1911. 


Action  of  the  Spermatozoon  upon  the  Egg       231 

Godlewski^  found  recently  that  the  eggs  of  sea-urchins  can 
be  fertiHzed  with  the  sperm  of  Chaetopterus,  an  annehd.  All  the 
eggs  form  the  fertilization  membrane,  bat  the}^  sooner  or  later 
begin  to  disintegrate  without  segmenting.  When  he  submitted 
such  eggs  to  hypertonic  sea- water  for  22  minutes,  they  developed 
into  larvae.  The  eggs  behave  as  if  the  foreign  sperm  had  only 
acted  through  the  membrane-forming  agency. 

The  interesting  fact  about  these  experiments  was  that  the 
spermatozoa  entered  the  eggs  and  even  fused  with  the  egg 
nucleus.  The  eggs  therefore  received  the  second  factor  con- 
tained in  the  spermatozoon,  and  3^et  they  did  not  develop. 
This  seems  to  indicate  that  this  second  factor  carried  by  the 
spermatozoon  is  much  more  specific  than  the  first  membrane- 
forming  factor.  This  specificity  is  perhaps  also  the  reason 
that  the  sea-urchin  eggs  fertilized  with  starfish  or  an}^  other 
foreign  sperm  die  in  such  large  numbers  in  the  gastrula  stage. 

i  Godlewski,  Archiv  f.  Entwicklungamechanik,  XXXIII,  196,  1911, 


XXIII 
THE  ACTION  OF  THE  SPERMATOZOON  UPON  THE  EGG 

II.      THE    COMBINATION    OF    ARTIFICIAL    PARTHENOGENESIS    AND 
FERTILIZATION    WITH    SPERM    IN    THE    SAME    EGG 

1.  Two  possibilities  exist  for  the  explanation  of  the  activa- 
tion of  the  egg  by  a  spermatozoon:  either  the  spermatozoon 
carries  a  ferment  or  a  catalyzer  into  the  egg  which  accelerates 
the  rate  of  the  chemical  reactions  in  the  egg;  or  it  removes  an 
obstacle  to  the  development.  The  role  of  the  membrane  forma- 
tion favors  the  latter  idea,  since  it  is  not  conceivable  that  all 
the  diverse  means  by  which  development  can  be  induced  act 
as  ferments;  moreover,  they  only  act  provided  they  cause  the 
membrane  formation.  Hence  the  membrane  formation  is  the 
essential  factor  which  induces  development  at  least  in  the  sea- 
urchin  egg.  This  makes  it  rather  improbable  that  the  sperma- 
tozoon induces  development  by  carrying  a  catalyzer  into  the 
egg  (although  it  may  carry  enzymes  for  other  purposes,  e.g., 

heredity) . 

This  idea  is  supported  by  other  facts.  We  know  that  the 
velocity  of  chemical  reactions  is  increased  if  the  quantity  of 
the  catalyzer  is  increased;  and  that  if  we  double  the  quantity 
of  the  catalyzer  the  rate  of  velocity  is  increased  in  the  ratio 
of  either  1:2  or  1 :  t/2.  The  rate  of  chemical  reactions  during 
development  can  be  measured  by  the  rate  of  cell  division  of  the 
egg,  as  is  evidenced  through  the  influence  of  temperature  upon 
the  rate  of  development  (chap.  iii).  Hence  if  the  spermatozoon 
caused  development  by  carrying  a  catalyzer  into  the  egg,  the 
rate  of  segmentation  should  be  either  twice  as  fast  or  1 .4  times 
as  fast  if  two  spermatozoa  enter  the  egg  as  if  only  one  sper- 
matozoon enters.  As  is  well  known,  cases  occur  in  which  two 
spermatozoa  enter  the  egg.     In  such  cases  the  egg  divides  into 

233 


234     Artificial  Parthenogenesis  and  Fertilization 

three  or  four  cells  instead  of  into  two.  The  writer  has  measured 
the  rate  of  segmentation  in  these  eggs  and  found  that  the  inter- 
val between  addition  of  sperm  and  cleavage  is  identical  with 
that  of  eggs  fertilized  by  only  one  sperm.  This  proves  that 
the  sperm  induces  development,  either  by  removing  an  obstacle 
e.g.,  a  substance  which  inhibits  development,  or  by  activating 
a  substance  contained  in  the  cortical  layer  which  was  inactive 
before  and  which  is  needed  for  development.  The  obstacle 
which  inhibits  development  is  obviously  the  state  and  constitu- 
tion of  the  cortical  layer  of  the  unfertilized  egg.  The  cytolysis 
or  destruction  of  this  layer  (which  results  in  membrane  for- 
mation) allows  the  egg  to  develop. 

Another  set  of  experiments  confirms  this  view.  It  is  con- 
ceivable that  the  fatty  acids  or  alkalies  by  which  we  call  forth 
the  membrane  formation  might  act  as  catalyzers.  If  that 
were  the  case,  the  superposition  of  the  fertilization  of  the  egg 
by  sperm  and  by  a  treatment  with  a  fatty  acid  should  accelerate 
the  rate  of  development  in  such  an  egg.  If  eggs  are  first  ferti- 
lized by  sperm  and  then  treated  with  butyric  acid  for  that 
length  of  time  which  is  required  for  artificial  membrane  forma- 
tion, no  acceleration  of  the  rate  of  cell  division  is  observed.  If 
we  call  forth  artificial  membrane  formation  first  by  butyric 
acid,  no  spermatozoon  can  enter  the  egg  since  the  fertilization 
membrane  is  impermeable  to  a  spermatozoon.  But  we  can 
destroy  the  membrane  by  shaking  it.  If  we  then  add  sperm 
to  such  eggs,  the  spermatozoa  enter,  cause  a  second  membrane 
formation  (in  which  the  membrane  fits  tightly  around  the  egg), 
and  the  eggs  develop  at  room  temperature  without  requiring 
any  further  treatment  with  the  hypertonic  solution;  which 
indicates  that  the  spermatozoa  have  entered  the  egg.  In 
such  eggs  the  rate  of  cell  division  is  exactly  the  same  as  in 
normally  fertilized  eggs.^ 

1  The  idea  that  the  spermatozoon  does  not  induce  development  by  carrying 
a  catalyzer  into  the  egg  was  set  forth  in  The  Dynamics  of  Living  Matter,  1906. 


Action  of  the  Spermatozoon  upon  the  Egg       235 


Finally,  the  rate  of  segmentation  is  the  same  in  the  eggs 
developing  parthenogenetically  as  in  eggs  fertilized  by  sperm. 
This  also  proves  that  the  sperm  does  not  induce  development 
by  any  catalytic  influence,  but  by  the  removal  of  an  obstacle 
or  an  inhibiting  factor  which  obviously  exists  in  the  condition 
of  the  cortical  layer.  Formerly  the  writer  had  suggested  that 
the  removal  of  this  obstacle  consisted  in  the  secretion  of  an 
inhibitive  substance  from  the  egg,^  a  view  which  Bataillon- 
and  Lillie^  have  since  adopted;  but  the  fact  that  complete 
cytolysis  of  the  unfertilized  sea-urchin  egg  by  saponin  raises 
the  rate  of  oxidations  in  the  same  way  as  membrane  formation 
or  fertilization  suggests  that  the  cytolysis  of  the  cortical  layer 
is  the  essential  removal  of  the  ''obstacle.'"* 

The  destruction  of  this  cortical  layer,  the  artificial  membrane 
formation,  leads  to  a  rapid  increase  in  the  rate  of  oxidations 
in  the  egg  of  the  sea-urchin.  These  oxidations  form  the  foun- 
dation of  all  the  further  cytological  changes  in  the  egg,  since 
their  suppression  inhibits  these  cytological  changes.  It  is, 
therefore,  obvious  that  the  point  which  demands  further  ex- 
planation is  the  connection  between  membrane  formation  or 
cytolysis  and  rate  of  oxidation.  It  is  conceivable  that  the  cor- 
tical layer  of  the  unfertilized  egg  forms  a  crust  impermeable 
to  oxygen,  but  there  is  no  proof  for  such  an  assumption.  It 
is  also  conceivable  that  there  is  present  in  the  surface  of  the 
egg  a  substance  which  inhibits  the  development  of  the  egg,  and 
that  this  substance  is  altered  or  removed  in  the  process  of 
membrane  formation.     It  is  finally  conceivable  that  the  surface 

»  Loeb,    University  of  California   Publications,  Physiology,   II,   1905. 

-  Bataillon,  "  Le  probleme  de  la  fecondation  circonscrit  par  rimpregnatlon 
sans  amphionyxie  et  la  parthenogenese  traumatique,"  Arch,  de  Zool.  expcr.  et 
grn.,  XLVI,  101,  1910. 

3  F.  Lillie,  Jour.    Morphol.,  XXII,  361,   1911. 

*  There  is  a  possibility  that  the  egg  contains  in  the  cortical  layer  a  catalyzer 
or  substances  caasing  an  increase  in  the  rate  of  chemical  reactions  in  the  egg. 
While  in  the  unfertilized  eggs  these  substances  are  not  able  to  act,  they  are  rendered 
available  if  the  cortical  layer  is  cytolyzed.  This  possibility  was  set  forth  by  me  in 
Proc.  Sac.  for  Exper.  Biol,  and  Med.,  VII,  No.  4,  April  20,  1910. 


236     Artificial  Parthenogenesis  and  Fertilization 


\siyer  of  the  unfertilized  egg  contains  a  substance  which  is 
needed  for  development  but  which  is  not  available  until  the 
surface  Lav^er  is  cytolyzed  or  destroyed  otherwise.  A  further 
discussion  of  these  possibilities  with  our  present  knowledge  of 
the  chemistry  of  the  egg  is  futile. 

2.  We  have  seen  that  the  spermatozoon  induces  develop- 
ment by  two  different  agencies,  one  of  which  has  a  membrane- 
forming  effect,  while  the  other  must  act  somewhat  like  the 
hypertonic  solution  in  our  method  of  artificial  parthenogenesis. 
We  stated  that  the  hypertonic  solution  has  merely  a  corrective 
effect  since  the  membrane  formation  sets  the  whole  machinery 
of  cell  division  into  action;  Boveri  suggested  that  the  spermato- 
zoon carries  in  its  middle  piece  the  centrosome,  the  real  organ 
for  cell  division,  into  the  egg. 

The  idea  that  the  centrosome  is  the  middle  piece  of  the 
spermatozoon  and  that  the  carrying  of  this  middle  piece  is  the 
main  function  of  the  spermatozoon  in  inducing  development 
does  not  agree  with  the  observations.  F.  Lillie^  points  out  that 
the  middle  piece  is  probably  not  carried  into  the  egg  at  all,  and 
he  proves  that  in  Nereis  any  piece  of  the  spermatozoon  is  able 
to  give  rise  to  centrosome  and  aster  formation.  These  forma- 
tions arise  in  the  egg  cytoplasm  which  is  in  contact  with  the 
sperm  fragment.  The  centrosome  and  aster  formations  are 
physicochemical  effects  induced  through  the  influence  of  the 
sperm  fragment.  Such  effects  are  also  induced  by  the  method 
of  artificial  parthenogenesis. 

Morgan^  found  that  supernumerary  astrospheres  may  arise 
if  fertilized  eggs  are  put  into  hypertonic  sea-water,  but  the  writer 
is  of  the  opinion  that  this  happens  only  if  the  eggs  remain 
too  long  in  the  hypertonic  solution.  Yet  it  was  natural  to 
consider  the  possibility  that  the  second  factor  which  the  sperma- 
tozoon must  supply  for  development  might  be  the  centrosome; 

1  F.  R.  Lillie,  "Studies  in  Fertilization,"  III  and  IV,  Jour.  Exper.  Zool,  XII, 
413.    1912.  • 

2  Morgan,    Archiv  f.    Entwicklungsmechanik,  VIII,   448,    1899. 


Action  of  the  Spermatozoon  upon  the  Egg       237 

and  that  the  second  treatment  with  the  hypertonic  solution 
might  only  be  needed  to  create  a  centrosome  de  novo  in  the 
egg.  The  idea  was  not  probable,  since  we  saw  that  the  mem- 
brane formation  alone  suffices  to  provide  the  egg  with  the  centro- 
somes  and  astrospheres  necessary  for  cell  division,  as  the  egg 
is  able  to  segment  if  the  temperature  is  not  too  high ;  and  sec- 
ond that  it  is  possible  to  substitute  for  the  hypertonic  solution 
the  suppression  of  oxidations,  a  factor  which  directly  sup- 
presses the  production  of  astrospheres.  Moreover,  the  experi- 
ments by  Hindle  have  shown  that  the  centrosomes  are  not 
formed  while  the  eggs  are  in  the  hypertonic  sea-water.  The 
following  observations  and  experiments  by  the  writer  indicate 
that  the  hypertonic  solution  does  not  act  in  these  experiments 
by  the  creation  of  astrospheres  or  centrosomes. 

When  we  put  the  unfertilized  eggs  of  S.  purpuratus  directly 
into  hypertonic  sea-water  (without  submitting  them  to  the 
butyric-acid  treatment)  and  if  we  put  them  back  at  different 
intervals  into  normal  sea-water,  we  find  that  if  the  eggs  have 
been  exposed  a  sufficiently  long  time  (two  hours  or  more)  to 
the  hypertonic  sea-water  a  number  will  begin  to  segment. 
These  eggs  will  often  go  only  into  the  two-  or  four-cell  stage, 
or  sometimes  to  the  eight-  or  sixteen-cell  stage,  and  then  stop 
developing.  They  cease  to  divide,  and  remain  in  the  resting 
stage. ^  Such  eggs  remain  after  this  perfectly  normal  and  they 
have  the  appearance  of  small  unfertilized  eggs.  If  we  wait 
for  some  time,  say  twxnty-four  hours,  to  make  sure  that  they 
neither  develop  nor  disintegrate,  and  add  sperm,  each  one  of 
these  blastomeres  forms  a  tightly  fitting  membrane.  They 
begin  to  develop  in  a  perfectly  normal  way  and  into  normal 
larvae.  We  are  then  dealing  with  eggs  which,  after  having 
been  treated  with  hypertonic  sea-water,  were  in  possession  of 
the  whole  apparatus  for  cell  division,  since  they  actualh'  had 

1  This  phenomenon  is  much  more  common  in  the  eggs  of  S.  purpuratus  than 
in  those  of  Arbacia. 


238     Artificial  Parthenogenesis  and  Fertilization 


segmented,  not  only  once,  but  many  times.  Why  did  they 
stop  developing?  Surely  not  for  lack  of  centrosomes,  since 
the  fact  that  they  segmented  showed  that  they  possessed  them. 
Our  experiment  therefore  proves  that  the  presence  of  astro- 
spheres  and  centrosomes,  and  their  ability  to  function,  does  not 
guarantee  the  possibility  of  development.^ 

But  it  is  not  necessary  to  fertilize  such  eggs  with  sperm; 
it  suffices  to  induce  membrane  formation  with  butyric  acid  and 
they  will  develop.     The  artificial  membrane  formation  will  have 


Fig.  74  Fig.  75 

Figs.  73.  74.  and  75. — Fertilization  of  blastomeres  of  an  egg  with  .sperm.  The 
biastomere.s  liad  been  produced  on  the  previous  day  by  treating  the  egg  with  a 
hypertonic  solution.  They  had  gone  into  a  resting  condition  and  upon  the 
addition  of  sperm  formed  a  membrane  and  segmented  regularly  as  the  figures 
indicate.  Fig.  73  represents  a  dividing  intact  egg,  while  Figs.  74  and  75  originated 
from  a  blastomere  of  eggs  which  had  divided  into  two  or  four  cells  on  the  previous 
day. 

this  effect  twenty-four  hours  or  longer  after  the  eggs  have 
been  treated  with  the  hypertonic  solution.  The  membrane 
fits  rather  tightly  in  this  case.  Hence  eggs  which  had  been 
caused  to  develop  b}^  treating  them  for  two  and  a  half  hours 
with  hypertonic  sea-water  and  which  had  stopped  segmenting 
for  twenty-four  hours  after  reaching  the  two-  or  four-cell  stage, 
can  be  caused  to  segment  regularly  and  develop  into  plutei  if 
they  are  treated  with  butyric  acid^  (Figs.  73-75). 

This  gives  the  impression  that  the  stoppage  of  the  develop- 
ment after  one  or  more  cell  divisions  had  been  caused  by  the 

1  Loeb,  "Die  Superposition  von  kiinstlicher  Parthenogenese  und  Samenbe- 
fruchtung  in  demselben  Ei,"  Archiv  f.  Entwicklungsmechanik,  XXIII,  479,  1907. 
-  Loeb,  Jour.  Exper.  Zool.,  XV,  201,  1913. 


Action  of  the  Spermatozoon  upon  the  Egg       239 


formation  of  a  new  cortical  layer  like  the  one  which  surrounds 
the  unfertilized  egg.  But  the  effect  of  the  previous  treatment 
of  the  egg  with  hypertonic  solution  must  have  lasted,  since 
otherwise  the  mere  membrane  formation  by  butyric  acid  in  these 
blastomeres  would  have  started  a  new  development,  but  would 
also  have  caused  the  rapid  disintegration  of  these  eggs.  What 
was  the  irreversible  effect  of  the  treatment  with  hypertonic 
sea-water?  Certainly  not  the  presence  of  centrosomes  and 
astrospheres,  since  in  the  next  paragraph  we  shall  show  that  if 
the  parthenogenetic  eggs  are  fertilized  while  the}-  are  still  in 
possession  of  centrosomes  or  astrospheres,  they  perish.  On  the 
contrary  these  eggs  which  developed  normally  after  artificial 
membrane  formation  had  lost  the  centrosomes  and  astrospheres 
the}'  had  possessed  immediately  after  the  treatment  with  the 
hypertonic  solution. 

We  have  stated  in  a  previous  chapter  that  the  rate  of  oxida- 
tions is  increased  six  times  by  a  spermatozoon.  In  eggs  treated 
for  two  and  a  half  hours  with  hypertonic  sea-water  the  rate  of 
oxidations  is  increased  less,  often  only  about  2.6  times,  and 
the  increase  is  apparently  not  exactly  the  same  in  the  eggs  of 
different  females.  We  noticed  also  that  after  some  time  the 
rate  of  oxidation  decreases.  Could  it  be  possible  that  the 
cessation  of  segmentation  is  due  to  the  fact  that  the  rate  of 
oxidation,  which  at  the  beginning  was  rather  low,  falls  below 
the  minimum  limit,  and  that  this  causes  the  standstill  of 
development  ?  This  standstill,  if  prolonged,  would  lead  to 
the  loss  of  the  centrosomes  (and  astrospheres),  just  as  these 
organs  are  lost  in  the  sea-urchin  egg  after  the  maturation 
division. 

This  leaves  one  point  still  unexplained,  namely,  the  irre- 
versible after-effect  of  the  treatment  with  the  hypertonic  solu- 
tion. As  we  stated  in  a  previous  chapter,  this  after-effect 
may  consist  in  the  fact  that  the  treatment  with  a  hypertonic 
solution  leads  to  the  formation  of  a  substance  which  remains  in 


240    Artificial  Parthenogenesis  and  Fertilization 

the  egg  permanently  and  which  saves  it  from  the  disintegra- 
tion which  follows  membrane  formation. 

3.  In  the  preceding  experiment  blastomeres  were  fertilized 
which  had  ceased  to  segment  for  about  twenty  hours.  The 
experiment  leads,  however,  to  an  entirely  different  result  if 
sperm  is  added  while  the  blastomeres  are  in  active  partheno- 
genetic  cell  division.  If  we  add  sperm  to  such  eggs  {S.  purpura- 
tus),  they  form  a  fertilization  membrane,  but  they  do  not 
develop  very  far.  The  entrance  of  a  spermatozoon  into  the 
blastomere  of  an  egg  which  is  in  active  parthenogenetic  seg- 
mentation leads  to  the  rapid  disintegration  of  the  egg  or  l)las- 
tomere;  while  the  entrance  of  a  spermatozoon  into  a  partheno- 
genetic blastomere  which  has  gone  back  into  the  resting  stage 
for  some  time,  can  cause  the  development  of  the  blastomere 
into  a  normal  pluteus.  What  causes  this  difference  ?  Possibly 
the  fact  that  the  blastomere  which  had  gone  back  into  the  rest- 
ing stage  for  some  time  has  lost  centrosomes  and  astrospheres, 
while  the  egg  which  is  in  active  parthenogenetic  cell  division 
possesses  both  organs. 

These  blastomeres  in  which  fertilization  by  sperm  is  super- 
imposed upon  artificial  parthenogenesis  while  the  eggs  are 
still  in  active  development  behave  like  eggs  fertilized  by  more 
than  one  spermatozoon.  Driesch  found  that  eggs  which  had 
been  fertilized  by  more  than  one  spermatozoon  do  not,  for  the 
most  part,  develop  beyond  the  blastula  stage.^  Boveri  has 
explained  this  by  the  fact  that  such  an  egg  possesses  more 
than  two  astrospheres.^  As  we  know,  the  division  of  the 
nucleus  into  two  daughter  nuclei  depends  upon  the  fact  that 
the  dividing  egg  forms  two  astrospheres.  This  is  the  case 
not  only  in  fertilization  by  sperm,  but  also  in  the  development 
started   by   the   methods   of   artificial   parthenogenesis.     But 

1  Driesch,     "  Ueber    die    Furchung    doppelbefruchteter    Eier,"     Zeitschr.    f. 
wissenschft.   Zool.,   LV,   1892. 

2  Boveri,  Zellenstudien,  Heft  6;    Die  Entwicklung  dispermer  Seeigeleier,  Leipzig, 
1907. 


Action  of  the  Spermatozoon  upon  the  Egg       241 


if  two  spermatozoa  enter  the  egg,  not  only  two  but  three  or 
four  astrospheres  are  formed.  When  we  cause  the  segmentation 
of  an  unfertilized  egg  by  treating  it  with  hypertonic  sea-water, 
two  astrospheres  are  formed  (unless  the  eggs  have  been  too 
long  exposed),  and  the  division  of  the  nucleus  takes  its  regular 
course.  But  if  the  eggs  are  left  too  long  in  the  hypertonic 
solution,  they  divide  into  more  than  two  cells  at  once  when  they 
are  put  back  into  normal  sea-water.  According  to  Morgan, 
this  is  owing  to  the  formation  of  more  than  two  astrospheres. 
Such  eggs  do  not  develop  into  normal  larvae. 

Why  is  it,  then,  that  only  those  eggs  develop  into  vigorous 
larvae  in  which  the  first  division  leads  to  the  formation  of  only 
two  cells  ?     To  this  question  Boveri  gives  the  following  answer. 
In  normal  nuclear  division,  each  chromosome  sphts  length- 
wise into  two  similar  pieces,  one  of  which  goes  into  each  of  two 
astrospheres,  and  into  the  new  nucleus;  so  that,  therefore,  after 
the  division  is  accomplished,  each  of  the  two  daughter  nuclei 
contains,  quantitatively  and  qualitatively,  the  same  nuclear 
material.     But  if  two  spermatozoa  enter  an  egg,  then  three  or 
four  astrospheres  are  formed,  and,  correspondingly,  three  or 
four  daughter  cells.     But  since  each  chromosome  of  the  mother 
nucleus  divides  only  into  two  parts,  it  is  naturally  impossible 
that  in  this  case  each  daughter  nucleus  will  contain  a  half  of 
each  chromosome  of  the  mother  nucleus.     Boveri  and  many 
other  authors  assume,  and  with  good  reason,  that  the  different 
chromosomes   of    the   nucleus   are   physiologically   dissimilar. 
It  will  therefore  be  apparent  that  equivalent  and  fully  potent 
daughter  nuclei  will  accordingly  result  only  from  regular  nuclear 
division  with  two  astrospheres;    and  that  when  three  or  more 
astrospheres  are  present  the  single  daughter  nuclei  will  not  con- 
tain the  full  number  and,  as  a  rule,  not  qualitatively  the  same 

nuclear  material. 

This  hypothesis,  then,  would  also  explain  why,  under  cer- 
tain conditions,  the  superposition  of  artificial  parthenogenesis 


242     Artificial  Parthenogenesis  and  Fertilization 

and  fertilization  with  sperm  curtails  the  life  of  the  larvae,  and 
prevents  them  from  reaching  the  pluteus  stage.  For  if  we  start 
the  eggs  developing  by  the  old  osmotic  method,  i.e.,  with 
gelatinous  membrane  formation,  and  then  fertilize  a  cell  of 
the  two-  or  four-cell  stage  with  sperm  while  they  are  still  ready 
to  segment,  the  next  division  of  each  of  these  blastomeres  w411 
lead  to  the  formation  not  of  two  cells,  but  of  three  or  four. 
To  the  astrospheres  which  are  already  formed  in  the  cell  by 
the  onset  of  parthenogenetic  development,  there  are  added 
also  the  astrospheres  formed  through  the  influence  of  the 
spermatozoon;  thus  the  next  division  of  the  nucleus  of  such  a 
cell  leads  to  the  formation  of  more  than  two  daughter  nuclei, 
which  are  also  usually  qualitatively  unlike.  They  are  there- 
fore in  the  position  of  an  egg  fertilized  by  two  spermatozoa, 
and  so  have  the  same  restricted  vitality  as  is  possessed  b}"  such 
eggs.  It  is  possible,  however,  that  fragments  which  are  ferti- 
lized by  sperm  while  they  are  in  the  process  of  partheno- 
genetic cell  division  suffer  more  than  normal  eggs  fertilized  by 
two  spermatozoa. 


XXIV 

CONDITIONS  FOR  THE  MATURATION  OF  THE  EGG 

1.  Before  we  discuss  experiments  upon  artificial  partheno- 
genesis in  other  forms  besides  sea-urchins,  we  must  examine 
the  phenomena  presented  by  the  ripening  egg.     We  mentioned 
in  the  introduction  that  development  proper,  i.e.,  the  segmenta- 
tion of  the  egg,  must  be  preceded  by  its  "maturation."     This 
is  a  process  which  consists  morphologically  in  the  reduction  of 
the  nucleus  by  two  divisions  and  the  extrusion  from  the  egg  of 
parts  of  the  nucleus  as  polar  bodies.     This  process  of  matura- 
tion shows  a  connection  with  the  process  of  fertilization  in  so 
far  as  in  many  forms  maturation  is  initiated  by  the  entry  of  a 
spermatozoon  into  the  egg.     In  other  forms  maturation  pro- 
ceeds spontaneously  either  in  the  ovary  (sea-urchin)  or  after  the 
eggs  have  been  shed  into  sea-water  (starfish),  and  until  this 
happens  the  spermatozoon  does  not  or  cannot  enter  the  egg. 
Whereas  one  finds  that  the  majority  or  very  many  of  the  eggs 
are  ripe  in  the  ovary  of  the  sea-urchin,  this  is  seldom  the  case 
with  the  starfish;   still  the  eggs  of  the  starfish  usually  mature 
quickly  on  being  put  into  sea-water.    When  the  eggs  of  the  star- 
fish are  removed  from  the  ovary,  they  possess  as  a  rule  large, 
conspicuous  nuclei.     Maturation  consists  in  the  reduction  of 
the  size  of  the  nucleus  by  a  double  division  and  the  extrusion 
of  the  polar  bodies.    The  time  that  elapses  before  these  processes 
take  place  in  sea-water  differs  for  the  eggs  of  different  starfish. 
This  probably  depends  on  the  fact  that  the  eggs  of  different 
females  are  not  all  in  the  same  condition  of  ripeness.     Experi- 
ments which  I  performed  a  few  years  ago  upon  the  maturation 
of  the  eggs  of  the  starfish  (Asterias  forbesii)  in  sea-water  indi- 
cated that  maturation  is  accelerated  by  two  of  the  substances 
present  in  sea-water,  viz.,  the  hydroxylions  and  the  oxygen.     If 

213 


244     Artificial  Parthenogenesis  and  Fertilization 


the  sea-water  is  neutralized,  or  slightly  acidified  by  the  addition 
of  acid,  maturation,  as  a  rule,  does  not  take  place,  although 
otherwise  it  occurs  with  rapidity.  If  the  eggs  of  the  same 
starfish  are  distributed  among  solutions  which  differ  only  in  the 
concentration  of  the  HO  ions,  it  will  be  found  that  the  rapidity 
with  which  maturation  occurs  increases  within  certain  limits 
with  the  concentration  of  the  HO  ions.  As  we  shall  see,  it 
is  possible  to  evoke  development  in  ripe  starfish  eggs  by  the 
use  of  acid;  hence  the  very  method  which  promotes  further 
development  in  mature  eggs  prevents  the  immature  egg  from 

maturing. 

The  writer  found  that  in  different  dishes  the  percentage 
of  matured  eggs  was  not  always  the  same  even  in  similar 
concentrations  of  hydroxj-lions.  This  indicated  the  existence 
of  another  factor  in  addition  to  the  concentration  of  the 
hydroxj^ions.  It  soon  turned  out  that  where  the  eggs  lay  in 
a  heap  maturation  proceeded  more  slowly  than  where  they  were 
spread  out  in  a  thin  layer.  This  gave  rise  to  the  suspicion  that 
the  supply  of  oxygen  might  be  important  for  the  maturation  of 
the  eggs.  Experiments  in  which  the  oxygen  of  the  sea-water 
was  driven  out  by  hydrogen,  as  well  as  those  in  which  KCN  had 
been  added  to  the  sea-water,  proved  that  in  these  cases  the  eggs 
failed  to  mature  in  spite  of  the  presence  of  the  hydroxy lions.^ 

2.  In  many  annelids  the  entrance  of  the  spermatozoon  into 
the  egg  causes  first  the  extrusion  of  the  polar  bodies,  and  after- 
ward development.  The  eggs  of  Polynoe,  however,  can  be 
induced  to  mature  even  in  sea-water  without  spermatozoa  by 
adding  to  the  sea-water  some  NaOH,  or,  better  still,  NH4OH, 
in  the  proportion  of  about  1.  5  c.c.  N/10  base  to  50  c.c.  of 
sea-water.  When  taken  out  of  the  animal  the  eggs  of  Polynoe 
are  of  irregular  shape;  they  are  surrounded  by  a  thick  chorion 
and  possess  a  large  nucleus.     In  ordinary  sea-w^ater  the  chorion 

1  Loeb,  "Ueber  Eireifung.  natiirlichen  Tod  iind  Verlangerung  des  Lebens 
beim  unbefruchteten  Seesternei,  usw.,"  Pfluger's  Archiv,  XCIII,  59,  1902;  Unter- 
sucliiingen,  p.  237. 


Maturation  of  the  Egg  245 


is  dissolved  or  liquefied  in  the  course  of  several  hours  (at  15°  C.) 
and  the  eggs  then  become  spherical.  The  maturation  of  the 
egg  begins,  but  is  not  completed,  as  the  polar  bodies  are  not 
extruded.  If  the  concentration  of  the  hydroxylions  in  sea- 
water  is  increased  (by  the  addition  of  NaOH  or  XH4OHJ  the 
polar  bodies  are  also  extruded  and  the  eggs  are  able  to  develop 
into  larvae.  But  if  no  alkali  is  added  to  the  sea-water 
the  eggs  go  to  pieces  in  the  course  of  the  next  twenty- 
four  hours  by  disintegrating  into  small  drops  or  fragments. 
From  these  experiments  it  follows  that  the  slight  concentration 
of  hydroxylions  present  in  sea-water  sets  in  action  the  matura- 
tion processes  in  the  egg,  but  not  enough  to  complete  this 
process  for  the  egg. 

If  a  trace  of  saponin  is  dissolved  in  sea-water  and  the  eggs  of 
Polynoe  (that  have  been  a  few  hours  in  sea-water)  are  placed  for 
one  minute  in  this  weak  saponin  solution,  they  form  a  perfect 
fertilization  membrane  and  in  the  course  of  from  five  to  thirty 
minutes  extrude  the  polar  bodies,  after  having  been  transferred 
to  normal  sea-water.  The  eggs  must,  however,  be  thoroughly 
washed  in  sea-water  to  remove  every  trace  of  saponin.  ^ 

In  the  egg  of  Chaetopterus,  another  annelid,  maturation  starts 
in  sea-water,  but  cannot  be  completed  unless  a  spermatozoon 
enters  the  egg.  As  has  already  been  noted,  Mead  observed 
that  the  addition  of  some  potassium  to  the  sea-water  brought 
maturation  to  completion. ^  In  Thalassema  also  the  sperma- 
tozoon enters  the  immature  egg  and  causes  both  the  extrusion 
of  the  polar  bodies  and  development.  Lefevre  found  that  the 
treatment  of  the  eggs  of  this  form  with  acid  caused  both  their 
maturation  and  development.^ 

1  Loeb,    "Ueber    die    Eiitwicklungserregung    unbefruchteter    Aiinrlideneier 
{Polynoe)  mittels  Saponin  und  Solanin,"  Pfluyers  Archiv,  CXXII,  448,  1908. 

'Mead,    Biological   Lectures  delivered  at    Woods  Hole,    189S   (Boston-  Uinn 
&  Co.). 

'Lefevre,    "Artificiai   Parthenogenesis   in    Thalassema  mellita."  Jour.  Exper 
Zool,  IV,  91,   1907. 


246     Artificial  Parthenogenesis  and  Fertilization 

It  is  a  remarkable  fact  that,  so  far  as  our  present  knowl- 
edge goes,  with  regard  to  those  eggs  in  which  the  entry  of  the 
spermatozoon  is  the  determining  factor  both  of  maturation 
and  development,  the  same  chemical  substances  that  will 
induce  artificial  maturation  also  induce  development;  whereas 
in  the  egg  of  the  starfish,  in  which  the  spermatozoon  does  not 
enter  until  after  maturation,  that  is  apparently  not  the  case. 

In  nature  everything  is  so  adjusted  that  when  the  eggs  are 
laid  they  are  (usually,  if  not  invariably)  susceptible  of  immediate 
fertilization.  Hence,  what  we  attain  in  these  experiments  by 
artificial  means  must  be  accomplished  by  the  organism  in  the 
natural  order  of  affairs.  I  had  started  to  investigate  these 
processes,  and  it  appeared  to  me  that  they  might  depend  on 
the  effect  of  the  blood  or  circulatory  fluid.  If  an  investigator 
obtains  by  chance  eggs  which  have  almost  matured  within  the 
organism,  he  cannot,  of  course,  fail  to  observe  that  they  are 
able  to  complete  the  maturation  process  without  the  aid  of  the 
processes  used  by  us.  But  it  would  be  wrong  to  follow  Mathews 
and  conclude  from  a  casual  observation  that  alkali  may  not  be 
necessary  for  maturation  because  he  observed  in  one  case  that 
(all?)  the  eggs  of  a  starfish  completed  their  maturation  in  a 
neutral  NaCl  solution.  There  is  nothing  surprising  in  this,  as 
in  starfish  the  eggs  of  the  same  female  show  different  degrees 
of  maturity,  in  that  a  few  mature  at  once  in  sea-water,  others 
slowly,  and  others  again  very  late,  or  perhaps  never;  while 
by  a  temporary  raising  of  the  concentration  of  the  hydroxylions 
the  maturation  generally  can  be  accelerated. 

Physiology  will  have  one  day  to  answer  the  question  why  in 
the  eggs  of  many  animals  the  spermatozoon  causes  both  matura- 
tion and  development,  whereas  in  other  cases  maturation  takes 
place  spontaneously,  the  egg  afterward  entering  into  a  resting 
condition,  out  of  which  it  is  aroused  only  by  fertilization.  In- 
vestigations upon  the  germination  of  oily  seeds  give  us  a  hint  as 
to  one  of  the  possibiUties  here  present.     For  if  some  substance 


Maturation  of  the  Egg  247 


such  as  an  acid  must  be  formed  before  the  one  or  more  enzymes 
necessary  for  development  can  be  activated,  we  can  under- 
stand that  the  absence  of  this  substance  must  lead  to  a  pause  in 
the  life-cycle  of  the  organism.  The  introduction  of  this  sub- 
stance from  without,  or  the  initiation  of  its  production  within, 
the  cell  will  then  effect  development. 

It  may  be  well  to  remember  also  in  this  connection  that 
the  unfertilized  eggs  of  purpuratus  can  be  induced  by  a  hy- 
pertonic solution  to  segment  into  two  or  four  cells,  without 
developing  further.  They  go  into  a  resting  stage  again,  from 
which  they  can  be  aroused  by  causing  artificial  membrane 
formation.  It  would  be  very  important  to  know  wh}^  these 
eggs  did  not  go  on  developing  after  they  had  started  to 
segment  under  the  influence  of  the  treatment  with  a  hyper- 
tonic solution. 

3.  It  appears  to  be  very  generally  the  case  that  maturation 
can  be  induced  by  treating  eggs  in  the  oocyte  stage  with  sodium 
hydrate;  for  I  have  obtained  results  similar  to  those  recorded 
for  Polynoe,  with  the  eggs  of  Nereis,  and  also  of  Sipuncidus. 
Unripe  eggs  which  refused  to  mature  in  sea-water  did  mature 
when  exposed  for  a  few  hours  to  hyperalkaline  sea-water  at  lo°C. 
With  regard  to  the  maturation  of  the  eggs  of  Nereis  at  Pacific 
Grove,  I  made  an  observation  that  may  perhaps  be  important 
from  the  point  of  view  of  the  mechanism  of  maturation  and 
membrane  formation.  In  the  immature  egg  of  Nereis  there 
is  a  greenish-blue  pigment  distributed  evenly  over  the  entire 
surface.  This  pigment  layer  contains  many  small,  highly  re- 
fracting droplets  which  may  be  fat  particles.  On  treating 
the  eggs  with  hyperalkaline  sea-water,  so  that  they  mature, 
the  following  changes  are  observed:  first,  a  membrane  is  lifted 
up,  comparable  to  the  fertilization  membrane  of  the  sea-urchin 
egg;  second — but  long  after  membrane  formation — the  numer- 
ous droplets  (fat?)  flow  together  into  a  few  large  drops;  third, 
the  greenish  mass,  which  had  previously  formed  an  even  layer 


248     Artificial  Parthenogenesis  and  Fertilization 


over  the  whole  egg,  contracts  and  gathers  the  fat-droplets  into 
one  hemisphere  of  the  egg.  Hence  there  arise  two  phases  on 
the  surface  of  the  egg,  one  of  which  apparently  contains  no 
fat  or  pigment,  while  the  other  obviously  contains  both. 

This  observation  led  me  to  consider  whether  the  importance 
of  fat-solvents  as  well  as,  in  part,  that  of  the  alkalies  at  the 
maturation  of  the  egg  ma}^  not  perhaps  consist  in  the  liquefac- 
tion of  solid  laj^ers  of  fat.  In  the  egg  of  Heteronereis  (of  Pacific 
Grove)  there  is  a  confluence  of  the  droplets  and  a  migration  of 
the  larger  drops  and  of  the  pigment  mass  to  one  hemisphere  of 
the  egg,  and  this  could  easily  be  explained  as  the  effect  of 
surface  tension.  There  is  obviously  an  analogy  between  the 
artificial  production  of  maturation  and  of  membrane  formation. 
In  both  cases,  the  process  of  solution  of  the  chorion  or  of  a 
substance  lying  at  the  surface  of  the  egg  appears  to  play  a  part. 


XXV 

ARTIFICIAL  PARTHENOGENESIS   IN  THE  EGGS  OF  THE 

STARFISH 

1.  The  experiments  on  artificial  parthenogenesis  in  starfish 
differ  in  an  essential  point  from  those  in  the  sea-urchin.  The 
sea-urchin  egg  undergoes  maturation  and  remains  at  a  state 
of  rest  while  in  the  ovary.  The  causation  of  development 
means,  therefore,  a  transition  from  the  resting  state  to  an  active 
state,  and  this  is  accompanied  by  a  rapid  increase  in  the  rate 
of  oxidations. 

The  conditions  in  the  starfish  egg  are  different.  When  these 
eggs  are  taken  out  of  the  ovary  they  are  as  a  rule  immature. 
As  soon  as  they  are  laid,  a  number  of  eggs,  which  varies  with 
the  individual  starfish,  begin  to  mature.  Not  until  one  or  both 
polar  bodies  are  thrown  out  can  a  spermatozoon  enter.  As 
soon  as  this  critical  stage  is  reached  the  egg  can  be  fertilized  by 
sperm.  If  it  is  not  fertilized  by  sperm  at  that  time  it  perishes 
in  a  few  hours.  There  is  then  this  difference  between  the  state 
of  the  sea-urchin  egg  and  that  of  the  starfish  egg  at  the  time 
of  fertilization:  The  starfish  egg  is  in  a  state  of  activity  since 
the  maturation  divisions  are  just  completed,  while  the  sea- 
urchin  egg  is  at  rest.  This  finds  its  expression  in  the  fact  that 
Wasteneys  and  I  found  that  the  entrance  of  a  spermatozoon 
into  the  starfish  egg  does  not  increase  the  rate  of  oxidations.^ 
It  harmonizes  with  this  result  that  the  writer  found  that  the 
process  of  maturation  of  the  starfish  egg  requires  conditions 
similar  to  those  for  the  development  of  the  sea-urchin  egg :  If 
oxygen  is  removed  from  the  sea-water  or  KCN  is  added  the  eggs 
remain  immature.  Moreover,  in  an  alkaline  solution  the  eggs 
ripen  more  rapidly  than  in  a  neutral  or  acid  solution. 

1  Loeb  and  Wasteneys,  Archiv  f.  Entwicklungsmechanik,  XXXV.  555,  1912. 

249 


250     Artificial  Parthenogenesis  and  Fertilization 


We  have  seen  that  the  spermatozoon  of  any  species  has 
substances  which  are  able  to  call  forth  the  development  of  the 
sea-urchin  egg.  It  harmonizes  with  this  fact  that  the  methods 
of  artificial  parthenogenesis  which  are  effective  in  the  sea-urchin 
must  also  be  effective  in  other  forms;  although  we  must  not 
forget  that  the  substances  used  in  artificial  parthenogenesis 
are  analogous  in  their  effects  but  not  necessarily  identical  with 
those  contained  in  the  spermatozoon.  This  explains  why  in 
each  form  quantitative  modifications  of  the  general  method 
are  required. 

We  shall  start  by  recounting  experiments  on  the  eggs  of 
the  Calif ornian  starfish  Asterina,^  the  eggs  of  which  form  a 
splendid  fertilization  membrane  after  the  entrance  of  a  sperma- 
tozoon. 

It  was  found  at  once  that  the  formation  of  this  membrane 
can  be  induced  by  the  same  agents  in  the  case  of  the  eggs  of 
Asterina  as  in  those  of  the  sea-urchin;  but  there  is  a  difference 
in  the  concentration  of  the  substances  required. 

When  the  mature  eggs  of  Asterina  are  placed  in  50  c.c.  of 
sea-water  in  which  1  c.c.  of  benzol  or  amylene  has  been  shaken 
up,  they  all  immediately  form  membranes  that  are  indistin- 
guishable in  appearance  from  those  formed  after  the  entrance 
of  a  spermatozoon.  If  the  eggs  are  not  removed  from  the  benzol 
or  amylene  sea-water  immediately  after  membrane  formation, 
they  succumb  to  cytolysis. 

If  one  of  the  lower  fatty  acids  is  used  instead  of  the  hydro- 
carbon, no  membranes  are  formed  so  long  as  the  Asterina  eggs 
remain  in  the  acidified  sea-water;  but  a  membrane  is  formed 
at  once  after  the  eggs  have  been  transferred  to  ordinary  sea- 
water,  provided  that  the  time  of  exposure  to  the  acid  has  been 
correctly  chosen.  Thus,  if  the  eggs  are  placed  for  two  minutes 
in  a  mixture  of  50  c.c.  of  sea-water+5  c.c.  N/10  acetic  acid, 

1  Loeb,  "Artificial  Membrane  Formation  and  Chemical  Fertilization  in  a 
Starfish  {Asterina),"  University  of  California  Publications,  Physiology,  II,  147, 
1905;    U ntersuchungen,  p.  349. 


Artificial  Parthenogenesis  in  Starfish  251 

they  form  beautiful  membranes  upon  transference  to  ordinary 
sea-water.  Butyric  and  caproic  acids  have  a  similar  effect, 
while  HCl  and  HNO3  have  much  less  or  even  no  effect. 

When  the  eggs  of  Asterina  are  removed  from  the  ovary,  they 
are,  as  we  have  seen,  immature,  i.e.,  they  possess  a  large  nucleus. 
These  eggs  cannot  be  fertilized  by  sperm;  it  is  also  impossible 
to  produce  membrane  formation  in  such  eggs  by  means  of  an 
acid.  Neither  fertilization  nor  artificial  membrane  formation  is 
possible  before  the  large  nucleus  has  broken  up  and  the  extrusion 
of  the  polar  bodies  has  started. 

After  artificial  membrane  formation,  the  starfish  eggs  begin 
to  divide.  But  in  this  they  exhibit  a  fundamental  difference 
in  their  behavior  from  sea-urchin  eggs  in  which  membrane 
formation  has  been  produced  by  a  fatty  acid.  For  whereas 
at  room  temperature  the  eggs  of  the  Californian  sea-urchin 
begin  to  disintegrate  after  mere  membrane  formation — unless 
they  have  been  treated  with  hypertonic  sea-water  or  with 
KCN — the  starfish  egg  is  better  off.  For  some  of  the  starfish 
eggs  that  have  formed  a  membrane  as  a  result  of  exposure  to 
butyric  acid  segment  regularly  and  develop  into  normal  larvae; 
though  the  rest  disintegrate,  like  sea-urchin  eggs,  after  mem- 
brane formation.  In  other  words,  the  starfish  eggs  differ 
from  those  of  the  sea-urchin  in  this,  that  they  do  not  depend 
upon  the  second  corrective  factor  with  the  same  degree  of 
necessity. 

In  one  experiment  the  eggs  of  an  Asterina  began  to  extrude 
their  polar  bodies  between  10:30  and  10:40.  Some  of  the  eggs 
were  then  fertihzed  with  sperm,  while  others  were  exposed  for 
one-half  to  one  and  a  half  minutes  to  the  action  of  6  c.c.  N/10 
butyric  acid +50  c.c.  of  sea-water.  In  both  lots  all  the  eggs, 
except  a  few  immature  ones,  formed  a  typical  fertilization 
membrane.  In  about  two  hours  all  the  sperm-fertilized  eggs 
entered  the  two-cell  stage,  and  at  about  the  same  time  some 
10  per  cent  of  the  eggs  treated  with  butyric  acid  also  began  to 


252     Artificial  Parthenogenesis  and  Fertilization 

divide.  Segmentation  proceeded  in  both  groups.  Five  hours 
after  the  butyric-acid  treatment  or  sperm  fertihzation,  respec- 
tively, the  position  was  as  follows:  The  eggs  fertilized  with 
sperm  were  all  in  the  sixteen-cell  stage.  Only  10  per  cent  of 
those  treated  with  butyric  acid  had  divided,  and  they  were  in 
the  eight-  to  sixteen-cell  stage.  The  others  had  not  divided 
and  showed  no  change.  But  soon  after  a  change  overtook 
them.  Small  clear  droplets  formed  irregularly  at  the  surface 
of  the  eggs  similar  to  those  extruded  from  sea-urchin  eggs  during 
heat  or  alcohol  cytolysis.  All  the  eggs  of  Asterina  which  formed 
these  drops  disintegrated.  Often  only  a  part  of  the  Asterina 
eggs  showed  this  formation  of  drops.  In  this  case,  only  those 
eggs  disintegrated  which  exhibited  this  formation  of  drops  on 
their  surface.  The  eggs  that  showed  no  drop  formation,  and 
segmented  normally  (i.e.,  about  10  per  cent  of  the  eggs  with 
butyric-acid  membrane),  developed  into  normal  larvae  just 
like  the  eggs  fertiHzed  with  sperm,  all  of  which  produced  normal 
larvae. 

These  experiments  are  very  interesting,  since  they  show 
that  a  not  inconsiderable  percentage  of  the  eggs  of  Asterina  will 
not  disintegrate  after  artificial  membrane  formation,  but  will 
develop.  These  eggs  therefore  behave  as  if  they  already  con- 
tained the  substance  or  structure  which  we  assume  is  formed 
under  the  influence  of  the  second  treatment.  We  may  perhaps 
be  justified  in  stating  that  in  Asterina  only  90  per  cent  of  the 
eggs  disintegrate  after  artificial  membrane  formation,  while 
10  per  cent  are  able  to  develop.  All  the  eggs  of  the  sea- 
urchins  {S.  purpuratus  and  Arbada)  disintegrate  after  artificial 
membrane  formation  unless  the}'  undergo  a  second  treatment. 
But  some  of  the  eggs  of  the  sea-urchins  at  Naples  seem  to  be 
able  to  develop  after  mere  artificial  membrane  formation. 

2.  Ralph  Lillie  in  working  on  the  eggs  of  a  starfish  of  the 
Atlantic  coast,  Asterias  forhesii,  found  that  a  short  heating 
of  the  eggs  to  between  35°  and  38°  C.  caused  them  to  form  a 


Artificial  Parthenogenesis  in  Starfish  253 


typical  fertilization  membrane.     The  length  of  exposure  neces- 
sary for  this  was  about  70  seconds  at  35°,  40  to  50  seconds 
at  36°,  about  30  seconds  at  37°,  and  about  20  seconds  at  38°. 
(These   eggs  can  withstand  a  higher  temperature  than    the 
eggs  of  S.   purpuratus,  which  are  killed   too  quickly  at  tem- 
peratures that  induce   membrane    formation    (34°   to   35°  C.] 
to  be  able  to  develop  subsequently.)     Lillie  observed  further 
that  some  of  the  starfish  eggs,  in  which  a  membrane  formation 
is  produced  by  rise  of  temperature,  develop  without  any  further 
treatment.     As  in  my  experiments  with  Asterina,  the  time  of 
membrane  formation  must  be  accurately  chosen;   for  the  time 
is  not  suitable  unless  the  eggs  are  ready  to  give  off  the  first  polar 
body. 

But  when  Lillie  put  the  eggs  after  artificial  membrane  forma- 
tion in  sea-water  to  which  enough  KCN  had  been  added  to 
make  it  about  a  N/2,000  solution  of  KCN,  many  more  eggs 
developed  than  in  cases  where  this  treatment  was  not  used.^ 
Now  this  is  exactly  the  same  result  that  I  obtained  with 
the  eggs  of  sea-urchins  after  artificial  membrane  formation 
(see  chap.  ix).  In  this  case  the  egg  was  given  more  time  to 
produce  the  second  factor  before  starting  on  its  development 
and  hence  more  eggs  survived. 

3.  My  first  experiments  with  starfish  were  carried  out  in 
1901,  when  Neilson  and  I  found  that  the  eggs  of  Asterias  for- 
besii  could  be  made  to  develop  when  maturation  had  taken  place 
by  putting  them  for  three  to  twenty  minutes  in  sea-water  to 
which  some  acid  had  been  added  (100  c.c.  of  sea-water -f 
3  to  5  c.c.  N/10  HCl  or  HNO3).'  If  the  eggs  were  then 
transferred  to  ordinary  sea-water,  some  of  them  began  to 
develop  into  larvae. 

* 

1  R.  S.  Lillie,  "Momentary  Elevation  of  Temperature  as  a  Means  of  Produ- 
cing Artificial  Parthenogenesis  in  Starfish  Eggs  and  the  Condition  of  Its  Action  " 
Jour.  Exper.  Zool.,  Y,  375,   1908. 

=  Loeb  and  Neilson,  Ffluger's  Archiv,  LXXXVII,  594,  1901;    Untersuchunaen 
p.  278. 


254     Artificial  Parthenogenesis  and  Fertilization 

Delage'  found  that  the  ripe  eggs  of  Asterias  glacialis 
developed  into  larvae  if  he  used  carbonic  acid  in  the  place  of 
the  acids  employed  by  us.  He  put  the  eggs  after  maturation 
in  sea-water  saturated  with  carbonic  acid  for  five  to  forty-five 
minutes.  As  shown  by  the  experiments  of  Godlewski  and 
myself,  carbonic  acid  is  one  of  the  acids  suitable  for  membrane 
formation;  it  diffuses  easily  into  the  egg  and  there  brings  about 
those  changes  which  underlie  membrane  formation.  The  greater 
efficiency  of  CO2  is  due  to  the  fact  that  it  is  a  very  weak 
acid  and  hence  diffuses  easily  into  the  egg. 

This  idea  is  supported  by  Delage's  observation  that  the 
number  of  Asterias  eggs  that  develop  after  the  carbonic-acid 
treatment  is  further  increased  if  the  oxygen  is  removed  for 
some  time  from  the  solution  (Delage,  1907),  though  he  does 
not  state  why  this  should  be  so.  We  can  understand  this  fact 
from  the  observation  which  the  writer  made  on  the  egg  of  the 
sea-urchin  that,  after  membrane  formation,  lack  of  oxygen 
gives  the  egg  an  opportunity  to  recover  from  the  threatening  dis- 
integration. The  same  thing  occurs  in  the  starfish  egg;  and 
we  can  understand  why  more  eggs  of  the  starfish  develop  if 
they  are  kept  without  oxj^gen  during  and  for  some  time  after 
the  exposure  to  CO2,  than  if  they  are  all  the  time  in  contact 
with  oxygen.  The  reason  for  this  is  to  be  found  in  the  fact 
that  treatment  with  CO2  causes  the  development  by  calling  forth 
the  change  in  the  cortical  layer  of  the  egg.  If  the  eggs  begin 
to  develop  under  these  conditions,  a  few  reach  the  larval  stage, 
while  many  disintegrate;  but  if  the  development  is  retarded 
for  a  while,  all  the  eggs  have  a  chance  to  recover  from  the 
threatening  disintegration  or  to  produce  the  substance  which 
saves  them  from  the  threatening  disintegration.  Unfortunately 
Delage  has  overlooked  my  experiments  upon  the  beneficial  effect 

1  Yves  Delage,  "Nouvelles  recherches  de  la  parthenogenese  experlmentale 
chez  Asterias  glacialis,"  Arch,  de  Zool.  expcr.  et  gen.,  3.  S.,  X,  213,  1902;  "  l^levage 
des  larves  parthenogenetiques  d' Asterias  glacialis,"  ibid.,  4.  S.,  II,  27,  1904; 
"  Nouvelles  experiences  de  parthenogenese  experimentale,"  ibid.,  4.  S.,  Ill,  CLXIV, 
1905;   Compt.  rend.  Acad.  Sc.  CXLV.  218,  1907. 


Artificial  Parthenogenesis  in  Starfish  255 

of  lack  of  oxygen  or  of  addition  of  KCN  upon  the  sea-urchin 
egg  after  membrane  formation,  and  hence  has  not  recognized 
that  his  results  harmonize  with  my  experiments. 

4.  Some  eggs  of  the  starfish  possess  a  certain  tendency  to 
develop  spontaneously  into  larvae  without  any  evident  external 
stimulus.  The  percentage  of  these  ''naturally"  partheno- 
genetic  eggs  fluctuates,  and  is  always  very  small.  It  is  also 
possible  that  this  tendency  toward  natural  parthenogenesis 
is  found  only  in  the  eggs  of  certain  females.  Mathews  dis- 
covered that  in  the  case  of  Asterias  forbesii  the  number  of  these 
eggs  that  reach  the  larval  stage  can  be  increased  by  shaking 
or  agitating  them.  The  amount  of  shaking  necessary  varies 
with  different  cultures:  .sometimes  a  very  vigorous  shaking 
in  a  test  tube  is  required,  at  others  the  mere  transference  of 
the  eggs  from  one  dish  to  another  by  means  of  a  pipette  is  suffi- 
cient. The  most  favorable  time  for  obtaining  this  result  is 
about  three  hours  after  the  liberation  of  the  eggs  from  the  ovary, 
probably  because  they  are  then  ripe. 

The  key  to  the  explanation  of  this  experiment  is  perhaps  to 
be  found  in  Mathews'  observation  that  these  eggs  after  being 
agitated  or  shaken  form  a  fertilization  membrane  and  then 
have  the  appearance  of  fertilized  eggs.^  Hence  we  must  regard 
this  membrane  formation  as  the  probable  impulse  to  develop- 
ment in  these  experiments.  But  how  can  membrane  formation 
be  produced  by  mere  agitation  ?  It  seems  to  me  that  this  can  be 
understood  if  we  suppose  that  it  depends  upon  the  destruction 
of  an  emulsion  in  the  cortical  layer  of  the  egg.  It  is  conceivable 
that  in  the  eggs  of  certain  forms  the  stability  of  this  emulsion 
is  so  small  that  mere  shaking  would  be  enough  to  destroy  it 
and  thus  induce  membrane  formation,  and  so  development. 

This  hypothesis  is  supported  by  an  observation  recently 
made  by  the  writer.  If  the  ovary  of  a  starfish  is  subjected  to 
moderate  pressure  and  the  eggs  are  afterward  removed,  it  is 

1  A.  p.  Mathews,  "Artificial  Partiienogenesis  Produced  by  Mechanical  Agita- 
tion," Am.  Jour.  Physiol.,  VI,  142,  1901. 


2o6     Artificial  Parthexogexesis  axd  Fertilization 


found  that  a  greater  or  smaller  number  of  them  have  formed 
membranes,  and  have  swelled  up  and  cytolyzed.  It  appears 
to  me  that  this  observation  can  be  explained  on  the  assumption 
that  the  pressure  destroys  in  the  egg  an  emulsion  that  was 
just  near  the  limits  of  stability. 

This  assumption  also  explains  why  occasionally  eggs  of  a 
starfish  may  develop  "spontaneously."  Such  eggs  probably 
form  a  fertilization  membrane  spontaneously.  The  case  is 
similar  to  the  spontaneous  segmentation  of  the  sea-urchin  egg, 
with  this  difference,  that  the  sea-urchin  egg  almost  always 
disintegrates  after  a  mere  membrane  formation,  while  some  of 
the  starfish  eggs  can  develop  without  a  second  treatment. 

Some  authors  have  stated  that  ''any  stimulus  induces  the 
egg  to  develop."  This  declaration  is  of  course  incorrect  for 
the  sea-urchin  egg,  and  no  authority  has  stated  it  for  this  egg; 
but  things  of  the  kind  have  been  credited  of  the  starfish  egg. 
The  statement  is  correct  within  the  limits  in  which  it  also  holds 
for  the  processes  of  cytolysis.  Cytolysis  can  be  induced  by 
very  different  agencies,  including  mechanical  disruption,  cer- 
tain chemicals,  heat,  and  high  and  low  concentrations  of  the 
solution.  The  reason  for  this  probably  consists  in  the  fact  that 
cytolysis  consists  in  the  destruction  of  an  emulsion,  and  that  this 
end  can  be  attained  by  very  different  methods.  It  is,  however, 
obviously  as  untrue  to  say  that  "any  stimulus  whatsoever" 
will  cause  the  eggs  of  the  starfish  to  develop,  as  it  would  be  to 
assert  that  every  stimulus  will  cause  c3'tolysis  or  haemolysis. 
It  is  needless  to  say  that  such  an  assertion  also  overlooks  the 
role  of  the  second  factor  in  the  causation  of  development. 

The  methods  of  artificial  parthenogenesis  for  the  egg  of  the 
starfish  resemble  those  of  the  sea-urchin  egg  very  closely.  The 
main  difference  between  the  two  types  of  eggs  exists  in  regard 
to  the  necessity  of  the  second  factor  of  fertilization,  which  does 
not  seem  to  be  required  in  the  case  of  a  small  percentage  of  the 
eggs  of  the  starfish. 


XXVI 

ARTIFICIAL  PARTHENOGENESIS   IN   THE   EGGS  OF 

ANNELIDS 

1.  The  eggs  of  the  anneUds  which  we  shall  consider  here 
differ  from  the  eggs  of  both  sea-urchin  and  starfish  in  that 
the  spermatozoon  enters  the  immature  egg  and  thus  sets  in 
motion  the  giving-off  of  the  polar  bodies  as  well  as  the  develop- 
ment of  the  egg.  In  the  case  of  the  starfish  the  spermatozoon 
must  enter  immediately  after  the  maturation  division  has 
begun  or  is  completed;  in  the  case  of  the  sea-urchin  the  egg 
can  remain  for  some  time  in  a  resting  condition  after  the  polar 
bodies  have  been  given  off. 

We  shall  first  consider  the  phenomena  of  fertilization  in 
Polynoe,  an  annelid  of  the  Pacific  coast.  When  the  eggs  are 
taken  out  from  the  animal  they  are  irregular  in  outline  and 
are  surrounded  by  a  thick  chorion.  The  large  nucleus  is 
plainly  visible.  When  the  unfertilized  eggs  are  put  into  sea- 
water  (which  contains  no  sperm)  the  chorion  is  dissolved  in  the 
sea-water  after  the  lapse  of  several  hours,  and  the  egg  then 
becomes  spherical  and  black.  Maturation  does  not,  however, 
proceed  farther  if  the  egg  remains  in  sea-water.  I  put  the 
eggs  of  Polynoe  into  sea-water  and  left  them  there  for  between 
two  and  four  hours  at  15°  C,  until  they  had  become  round. 
They  were  then  placed  in  a  watch  glass  with  4  c.c.  of  sea- 
water  to  which  had  been  added  15  drops  of  a  very  weak  solu- 
tion of  saponin  in  sea-water.  Now  the  eggs  which  had  been 
treated  with  saponin  for  one  minute,  and  then  washed,  formed 
a  beautiful  fertilization  membrane  and  began  to  extrude  their 
polar  bodies  in  from  five  to  thirty  minutes.  Some  of  the  eggs 
developed  into  swimming  larvae  in  the  course  of  from  eighteen 
to    twenty-four    hours,    in    most    cases   without     undergoing 

257 


258     Artificial  Parthenogenesis  and  Fertilization 


segmentation;  a  few  segmented  into  two  or  four  cells,  but  not 
farther.  One  often  saw  eggs  that  had  remained  stationary  in 
the  two-cell  stage,  swimming  about  as  larvae,  although  they 
always  consisted  of  merely  the  two  cells.  The  eggs  developed 
very  slowly  into  larvae  in  the  course  of  from  eighteen  to 
twenty-four  hours.  Fertilized  eggs  reached  the  trochophore 
stage  in  eight  hours  at  the  same  temperature.^  These  experi- 
ments in  which  the  eggs  of  Polynoe  are  made  to  develop  by 
merely  producing  membrane  formation  by  means  of  saponin 
(or  solanin)  are  not  invariably  successful,  and  the  number  of 
developing  larvae  was  often  very  small.  But  they  indicate 
that  the  development  of  the  egg  depends  upon  the  same 
conditions  as  in  the  sea-urchin  egg,  namely  upon  membrane 
formation. 

It  was  next  tried  whether  or  not  development  could  be  made 
more  normal  by  exposing  the  eggs  to  hypertonic  sea-water 
after  membrane  formation.  This  proved  to  be  the  case. 
Immature  eggs  freshly  taken  from  the  animal  were  subjected 
to  treatment  with  saponin.  Two  drops  of  a  very  weak  solution 
of  saponin  were  added  to  5  c.c.  of  sea-water,  and  after  four 
minutes  the  eggs  were  transferred  to  ordinary  sea-water  and 
freed  from  saponin  by  being  washed  four  times.  The  eggs  did 
not  form  a  membrane  in  the  saponin  solution,  but  the  chorion 
was  dissolved,  and  the  eggs  rounded  off.  They  were  then  put 
into  hypertonic  sea-water  (50  c.c.  of  sea-water H-8  c.c.  2^  m 
NaCl),  and  portions  of  the  eggs  were  replaced  in  ordinary  sea- 
water,  after  exposures  of  60,  104,  140,  162,  and  180  minutes 
respectively.  All  the  eggs  formed  fertilization  membranes 
in  the  hypertonic  solution;  this  was  a  secondary  effect  of  the 
exposure  to  saponin.  However,  the  eggs  placed  in  sea-water  as 
a  control  also  formed  a  membrane  there. 

The  control  eggs,  and  those  exposed  to  the  hypertonic 
sea-water  after  the  saponin  treatment  for  only  one  hour,  did 

1  Loeb,  "Ueber  die  Entwicklungserregung  iinbefruchteter  Annelideneier 
(Polynoe)  mittels  Saponin  und  Solanin,"  Pfluger's  Archiv,  CXXII,  448,  1908. 


Artificial  Parthenogenesis  in  Annelids  259 


not  divide,  and  did  not  in  this  experiment  develop  into  larvae. 
On  the  other  hand,  practically  all  of  the  eggs  treated  with 
saponin  that  had  been  two  hours  and  twenty  minutes  in  the 
hypertonic  sea-water  segmented,  but  reached  only  the  eight-cell 
stage  in  four  hours  after  removal  from  the  hypertonic  sea-water. 
Four  hours  later  these  eggs  had  reached  the  trochophore  stage 
and  were  swimming  about.     But  it  is  doubtful  whether  they 
had  undergone  any  further  segmentation.     Eggs  that  had  been 
exposed  only  to  hypertonic  sea-water,  without  previous  mem- 
brane formation  under  the  influence  of  saponin,  did  not  develop 
into  larvae.     This  experiment  was  repeated,  and  it  was  then 
found   that,  though    by   producing   membrane   formation   by 
means  of  saponin  a  few  eggs  of  Polynoe  can  indeed  be  made  to 
develop  into  larvae,  a  subsequent  exposure  to  hypertonic  sea- 
water  causes  many  more  eggs  to  develop.     The  rate  of  develop- 
ment is  also  increased  by  this  method.     The  analogy  in  behavior 
between  the  eggs  of  Polynoe  and  of  the  sea-urchin  is  obvious, 
although  not  complete,  since  the  sea-urchin  eggs  all  develop 
in  this  case  by  segmentation,  while  the  eggs  of  Polynoe  develop 
without  or  only  with  incomplete  segmentation. 

The  eggs  of  Polynoe  resemble  the  eggs  of  the  starfish,  in 
so  far  as  the  membrane  formation  suffices  for  the  causation  of 
development  of  some  eggs,  and  in  so  far  as  the  addition  of  the 
second  corrective  factor  increases  the  number  of  eggs  capable 
of  development. 

2.  There  is  yet  another  method  by  which  the  unfertilized 
eggs  of  Polynoe  can  be  made  to  develop.  This  is  by  raising  the 
alkalinity  of  the  sea-w^ater.^ 

If  the  concentration  of  the  hydroxylions  in  sea-water  be 
raised  by  the  addition  of  a  considerable  amount  of  sodium 
hydrate,  not  only  can  the  unfertilized  eggs  of  Polynoe  be  induced 
to  ripen,  but  a  small  percentage  of  them  will  segment  into  two 
or  four  cells;   and  sometimes  a  large  percentage  or  even  all  the 

1  Loeb,  "Ueber  die  allgemeinen  Mi;thoden  der  kiiustlichon  Parthonogeni'se.' 
Pfiiiyeri.    Archiv.   CXVIII,   572,    1907. 


260     Artificial  Parthenogenesis  and  Fertilization 

eggs  will  develop  into  larvae  without  segmentation.  In  one 
experiment  the  unfertilized  eggs  of  a  female  were  placed  in 
50  e.c.  of  sea-water +  1 . 5  c.c.  N/10  NaOH  and  left  for  some  tmie 
in  that  solution.  Four  hours  later  all  the  eggs  had  formed  a 
definite  membrane  and  extruded  both  polar  bodies.  After  eight 
hours  a  small  number  of  the  eggs  were  in  the  two-cell  stage — 
cleavage  was  quite  normal — but  the  rest  remained  undivided. 
Twenty-four  to  forty-eight  hours  after  the  start  of  the  experi- 
ment the  majority  of  the  eggs  had  developed  into  swimming 
larvae.  But,  externally,  segmentation  did  not  appear  to  have 
extended  beyond  the  two-  or  four-cell  stage.  The  control  eggs 
that  had  been  left  in  ordinary  sea-water  had  not  matured,  and 
in  the  course  of  twenty-four  hours  they  fell  to  pieces.  This 
oft -repeated  experiment  proves,  therefore,  that  the  unfertilized 
eggs  of  Polynoe  can  be  made  to  mature  and  develop  into  larvae 
by  exposing  them  for  some  time  to  hyperalkaline  sea-water. 

But  there  is  one  important  consideration  that  must  be  taken 
into  account,  and  that  is  a  favorable  supply  of  oxygen.  The 
eggs  developed  in  large  numbers  only  when  contained  in  lightly 
covered  watch  glasses,  while  the  eggs  left  in  the  big  dish  in 
which  they  were  separated  from  the  air  by  some  2  cm.  of  water 
matured  and  developed  in  much  smaller  quantities.  Perhaps, 
too,  the  neutralization  of  surplus  alkali  by  the  carbonic  acid 
of  the  air  is  effected  much  more  quickly  in  the  watch  glass 
than  in  the  larger  dish.  I  convinced  myself,  however,  that  if 
the  eggs  are  transferred  from  the  hyperalkaline  to  normal 
sea-water,  the  formation  of  larvae  occurs  only  if  the  eggs  have 
been  at  least  from  four  to  six  hours  in  hj^peralkaline  sea-water; 
and  even  then  the  results  are  much  worse  than  when  the  eggs 
remain  all  the  time  in  the  hyperalkaline  solution.  If,  however, 
the  treatment  with  a  hypertonic  solution  was  combined  with 
that  by  alkah,  less  alkali  could  be  used  and  more  eggs  segmented 
and  the  segmentation  advanced  farther  (especially  in  the  case 
of  weak  bases). 


Artificial  Parthenogenesis  in  Annelids  2(31 


It  was  easily  discovered  that,  as  in  the  egg  of  purpuratiis, 
a  neutral  hypertonic  solution  cannot  produce  development  in 
the  egg  of  Polynoe;  for  this  effect  is  possible  only  in  alkaline 
hypertonic  solution.  To  50  c.c.  of  a  neutral  m/2  van't  Hoff 
solution+9  c.c.  2^  m  NaCl  there  was  added  0.5  c.c.  N/IO 
NaOH;  another  such  hypertonic  solution  was  prepared  without 
the  alkali.  After  two  hours  in  these  solutions  the  eggs  were 
transferred  to  normal  sea-water.  Most  of  the  eggs  taken  from 
the  neutral  hypertonic  solution  formed  no  membrane  in  normal 
sea-water;  they  did  not  extrude  polar  bodies,  segment,  or 
develop.  On  the  other  hand,  about  1  per  cent  of  the  eggs  that 
had  been  in  the  alkaline  hypertonic  solution  segmented  per- 
fectly regularly  in  the  course  of  a  few  hours  as  far  as  the  eight- 
cell  stage,  and  the  majority  of  the  eggs  developed  into  swimming 
larvae.  The  development  of  the  eggs  was  generally  quicker 
than  in  the  case  of  unfertilized  eggs  exposed  to  hj-peralkaline 
but  not  hypertonic  sea-water. 

Again,  eggs  that  remained  between  two  and  six  hours  in 
the  neutral  hypertonic  solution  did  not  develop  when  subse- 
quently transferred  to  ordinary  sea-water.  But  when  the 
unfertilized  eggs  of  Polynoe  were  first  put  for  two  hours  into 
a  neutral  hypertonic  solution  and  then  for  not  more  than  four 
hours  into  50  c.c.  sea-water+0.5  c.c.  N/10  NaOH,  large  num- 
bers of  them  developed  into  swimming  larvae  upon  trans- 
ference to  normal  sea-water.  But  if  the  eggs  were  put  for 
four  hours  into  50  c.c.  sea-water -f  0.5  c.c.  N/10  NaOH  without 
being  exposed  to  the  hypertonic  solution,  as  a  rule  no  eggs, 
or  only  a  few,  developed.^ 

It  is  hardly  necessary  to  point  out  the  analogy  between  the 
effect  of  alkaline  and  hypertonic  sea-water  upon  the  eggs  of 
sea-urchins  and  of  Polynoe.  Like  saponin,  the  alkali  produces 
solution  of  the  chorion  and  membrane  formation  in  Polynoe. 

'  Loeb,  "  Ueber  die  allgemeinen  Methoden  der  kiinstlichen  Parthenogenese." 
Pfluger's  Archiv,  CXVIII,  572.  1907. 


262     Artificial  Parthenogenesis  and  Fertilization 

This  results  in  the  extrusion  of  the  polar  bodies  and  develop- 
ment. But  the  effect  of  the  alkaline  solution  is  enhanced  by 
the  treatment  of  the  eggs  with  hA^pertonic  sea-water. 

3.  We  have  already  stated  that  the  weak  bases  are  much 
more  efficient  in  causing  artificial  parthenogenesis  than  the 
strong  bases.  The  best  effects  were  produced  with  the  amines, 
especially  butylamine  and  benz^damine;  next  in  efficiency  were 
NH4OH  and  trimethylamine,  and  the  weakest  effects  were  pro- 
duced by  the  strong  bases  NaOH  and  tetraethylammonium- 
hj'droxide. 

To  give  an  example:  to  50  c.c.  hypertonic  sea-water  (100  c.c. 
sea-water+18  c.c.  2J  m  NaCl)  were  added  1.5  c.c.  N/10 
NaOH,  benz^damine,  and  NH4OH,  respectively.  Of  the  eggs 
that  had  been  forty-five  minutes  in  benzylamine,  40  per  cent 
developed;  the  same  result  was  produced  by  an  exposure  in 
NH4OH  of  80  minutes.  An  exposure  of  115  minutes  in  NaOH 
gave  a  much  smaller  number  of  larvae.  Those  treated  with 
NaOH  reached  the  swimming  stage  as  a  rule  much  later  than 
those  treated  with  an  amine.  The  eggs  treated  with  the  amine, 
especially  with  butylamine  and  benzylamine,  segmented  almost 
normally,  although  much  more  slowly  than  the  eggs  fertilized 
with  sperm. 

In  repeating  these  experiments  with  sea-water  made  alkaline 
(but  not  hypertonic),  the  writer  was  struck  with  the  fact 
that  the  eggs  developed  into  larvae  without  segmentation,  and 
very  slowly,  while  with  both  hypertonic  and  hyperalkaline 
sea-water  they  segmented  and  developed  more  rapidly  into 
larvae,  though  still  more  slowly  than  the  eggs  fertilized  by  sperm. 

It  is  obvious  that  when  the  hypertonic  solution  acts  in  this 
way  it  makes  the  development  more  normal.  This  may  also 
be  a  ''second  factor"  effect.  While  the  eggs  of  Polynoe  do 
not  directly  disintegrate  after  artificial  membrane  formation, 
they  show  their  abnormal  state  by  developing  without  segmen- 
tation.    The  hypertonic  solution  remedies  this  defect,  without, 


Artificial  Parthenogenesis  in  Annelids 


263 


however,  acting  as  completely  as  it  does  in  the  sea-urchin  egg 
after  membrane  formation. 

4.  Mead^  observed  that  the  addition  of  a  small  amount  of 
KCl  to  the  sea-water  causes  the  eggs  of  Chaetopterus  to  throw 
out  their  polar  bodies,  and  the  writer  observed  that  the  same 
procedure  causes  these  eggs  to  develop  into  larvae.^  I  had 
already  expressed  the  suspicion  that  these  eggs  develop  into 
larvae  without  segmentation.  F.  Lillie  made  sure  of  this  fact 
by  a  cytological  examination  of  the  eggs.^ 

All  efforts  to  cause  this  egg  to  undergo  development  with 
normal  segmentation  failed  until  Wasteneys  and  the  writer 
tried  the  effects  of  foreign  blood  serum.  The  eggs  were  put  for 
from  IJ  to  2i  minutes  into  a  mixture  of  25  c.c.  3/8  m  SrCl2  + 
25c.c.  m/2  (NaCH-KCi+CaCla),  then  for  10  minutes  into 
ox  serum  diluted  with  its  own  volume  of  m/2  Ringer  solution 
and  then  for  30  minutes  into  hypertonic  sea-water.  Such  eggs 
when  transferred  back  to  normal  sea-water  segmented  and  de- 
veloped into  larvae.^  They  were  usually  not  normal  larvae, 
inasmuch  as  they  showed  a  tendency  to  stick  to  the  glass  and 
to  each  other.  Moreover,  the  cleavage  cells  of  the  same  egg 
fell  apart  easily.     The  method  needs  further  improvement. 

5.  Lefevre  succeeded  in  causing  artificial  parthenogenesis 
in  the  eggs  of  Thalassema  mellita,  a  marine  annelid  of  North 
Carolina.^  The  eggs  of  this  worm  are  fertilized  in  the  oocyte 
stage,  just  like  those  of  Polynoe.  The  entry  of  the  spermato- 
zoon leads  to  the  formation  of  a  fertilization  membrane;  the 
two  polar  bodies  are  then  extruded,  and  segmentation  and  devel- 
opment start  after  maturation  is  complete.  Now  Lefevre 
found  that  when  the  unripe  eggs  of  Thalassema  are  exposed  to 

1  Mead,  Bioloijical  Lectures  delivered  at  Woods  Hole,  1898. 

2  Loeb,   Am.  Jour.  Physiol.,  IV,  423,   1901. 

3  F.  R.  Lillie,  Archiv  f.  Entwicklungsmechanik,  XIV,  477.  1902. 

"  Loeb  and  Wasteneys,  Science,  XXXVI,  255,  1912. 

5  G.  Lefevre,  "Artificial  Parthenogenesis  in  Thalassema  mellita,"  Jour.  Exper. 
Zool.,  IV,  91,   1907. 


264     Artificial  Parthenogenesis  and  Fertilization 


acid  (the  kind  of  acid  seems  immaterial)  ^  and  then  transferred 
to  sea-water,  they  form  membranes,  extrude  the  polar  bodies, 
and  often  develop  into  normal  larvae  after  a  perfectly  normal 
segmentation.  His  best  results  were  obtained  with  the  follow- 
ing mixtures  of  acids: 

17  CO.  N/10  HXO3+83  CO.  sea-water.     Length  of  exposure  5  minutes 
15  c.c.  N/10  HCl+So  CO.  sea-water.     Length  of  exposure  5  minutes 
10  c.c.  N/10  H2SO4+9O  c.c.  sea-water.     Length  of  exposure  8  minutes 
12  c.c.  N/10  Oxahc  acid-|-88  c.c.  sea-water.     Length  of  exposure  8 

minutes 
15  c.c.  N/10  Acetic  acid+85  c.c.  sea-water.     Length  of  exposure  5 

minutes 

Under  the  influence  of  acid  the  eggs  of  Thalassema  form  a 
typical  fertilization  membrane  after  transference  to  ordinary 
sea-water,  just  like  sea-urchin  eggs;  but  whereas  the  latter  must 
be  transferred  for  a  short  time  to  hypertonic  sea-water  after 
membrane  formation,  in  order  to  insure  their  development, 
this  is  not  necessary  with  the  eggs  of  Thalassema.  In  this 
respect  then  the  eggs  of  Thalassema  behave  like  those  of 
Asterma.  All  the  eggs  of  Thalassema  form  fertilization  mem- 
branes under  acid  treatment,  but  not  all  of  them  develop. 
In  the  most  propitious  circumstances  60  per  cent  of  the  eggs 
develop. 

The  velocity  of  maturation  and  of  the  onset  of  cleavage 
was  appreciably  less  than  in  the  case  of  the  fertilization  with 
sperm.  Whereas  in  the  fertilization  with  sperm  the  first  of  the 
polar  bodies  was  extruded  twenty  minutes  after  entry  of  the 
spermatozoon,  the  same  event  did  not  take  place,  when  the 
eggs  were  exposed  to  acid,  until  45  to  90  minutes  after  the  eggs 
had  been  transferred  from  the  acid  to  normal  sea-water.  The 
first  cell  division  took  place  in  50  to  60  minutes  after  the  entry 
of  the  sperm,  while  it  did  not  occur  until  two  to  three  and  a  half 
hours  after  the  acid  treatment  of  the  unfertilized  eggs. 

1  He  remarks  expressly  that  carbonic  acid  has  just  as  much  effect  as  any  other 
acid,  neither  more  nor  less. 


Artificial  Parthenogenesis  in  Annelids  265 

Lefevre  has  made  a  very  interesting  observation  on  the 
behavior  of  the  polar  bodies.     As  a  rule,  the  first  j^olar  bod}- 
given  off  from  the  egg  when  it  is  fertilized  with  sperm  divides 
only  once,  and  the  second  polar  body  does  not  divid(^     Lefevre 
observed,  however,  that  both  polar  bodies  of  the  eggs  treated 
with  acid  pass  through  a  series  of  mitotic  divisions  and  give 
rise  to  miniature  embryos  of  sixteen  cells.     He  compares  these 
facts  with  Francotte's  observation  that  in  Prostheceracus,  a 
turbellarian,  the  first  polar  body  is  relatively  large  and  can'be 
fertilized  by  a  spermatozoon  and  even  develop  into  a  gastrula. 
As  a  rule  the  cleavage  was  normal  and  so  were  the  larvae; 
but  they  did  not  rise  to  the  surface  of  the  water  like  those 
derived  from  fertilized  eggs.     Hence  they  easily  fall  victims  to 
bacteria. 

6.  For  the  sake  of  completeness,  some  of  the  older  experi- 
ments on  artificial  parthenogenesis  in  annelids  may  be  briefly 
mentioned,  although  the  methods  used  are  not  very  satisfactory. 
In  Amphitrite  eggs  could  be  caused  to  develop  by  treating  them 
with  sea-water  whose  calcium  content  was  raised.^ 

Fischer  produced  artificial  parthenogenesis  in  the  eggs  of 
Nereis  by  treating  them  with  a  hypertonic  solution.^  Bullot 
succeeded  by  the  same  method,  in  the  eggs  of  Ophelia.^ 

Again  we  may  state  that  the  methods  of  artificial  partheno- 
genesis for  annelids  are  essentially  identical  with  those  used  for 
the  eggs  of  the  sea-urchins,  except  that  the  second  factor  is 
not  quite  so  important. 

iLoeb.   Fischer,  and  Neilson,    Pfluger's   Archiv,  LXXXVII    1    1901-    ^mtt 
Jour.  Exper.   ZooL,  III,  49,   1906.  '      '  ' 

■'  Fischer,  Am.  Jour.  Physiol.,  IX,  100,  1903. 

3  Bullot,  Archiv  f.  Entwicklungsmechanik,  XVIII,  161,  1904. 


XXVII 

EXPERIMENTS  WITH  THE  EGGS  OF  MOLLUSCS 

In  1902  Kostaiiecki  succeeded  in  producing  the  early  seg- 
mentation stages  (two  to  four  cells)  in  the  unfertilized  eggs  of  a 
mollusc  (Madra),  by  exposing  them  for  two  hours  to  hypertonic 
sea-water.^  In  1903  the  writer  showed  that  the  unfertilized  eggs 
of  another  mollusc,  Lottia  gigantea,  and  of  several  forms  of 
Acmaea  can  be  made  to  develop  into  swimming  larvae  by 
treating  them  with  hypertonic  sea-water.^  The  method  consists, 
in  principle,  of  placing  the  eggs  for  two  hours  in  a  mixture 
of  50  c.c.  of  sea-water+10  c.c.  2J  m  NaCl.  The  number  of 
larvae  that  developed  was  always  small  (only  about  2  to  5 
per  cent  of  the  eggs),  and  their  vitality  was  low.  As  a  rule 
they  succumbed  after  thirty-six  to  forty-eight  hours.  After  I 
had  recognized  the  part  played  by  bases  in  the  activation  of  the 
eggs  of  the  sea-urchin,  it  next  occurred  to  me  to  find  out  whether 
the  osmotic  causation  of  development  in  Lottia  is  also  acceler- 
ated or  improved  if  this  hypertonic  solution  is  rendered  alkaline. 
This  was  found  to  be  true.  Thus  to  50  c.c.  of  (neutral)  m/2 
van't  Hoff's  solution+12  c.c.  of  2§  m  NaCl  were  added  in 
different  bowls  0,  0.1,  0.2,  0.4,  and  0.8  c.c.  N/10  NaOH. 
Among  these  solutions  the  eggs  of  a  Lottia  were  distributed 
after  being  washed  in  a  neutral  solution  (temperature  17.5°  C). 
Samples  of  the  eggs  were  transferred  to  normal  sea-water  after 
if,  2j,  2 J,  3|,  and  3f  hours  respectively.  The  eggs  exposed 
to  neutral  hypertonic  solution  did  not  develop,  and  the  same 
was  the   case   with  eggs   that   had  been  treated  with  0.1  c.c. 

'  Kostanecki,  "  Zytologische  Studien  an  kiinstlich  parthenogenetisch  sich 
entwickelnden  Eiern  von  Mactra,"  Arch.  f.  mikroscop.  Anat.  u.  E ntwicklungsgesch., 
LXIV.  1,  1904. 

2  Loeb,  University  of  California  Publications,  Physiology,  I,  7.  1903;  Untersuch- 
ungen  ueber  kiinstliche  Parthenogenese,  p.  283. 

267 


268    Artificial  Parthenogenesis  and  Fertilization 


NaOH.  Some  of  the  eggs  that  had  been  exposed  to  solutions 
containing  more  NaOH  did  develop  into  larvae;  the  percentage 
of  larvae  varied  according  to  the  alkalinity  of  the  solution  and 
the  time  the  egg  was  exposed  to  the  solution.  Many  of  the 
eggs  that  had  been  2j  hours  in  the  hypertonic  solution  con- 
taining 0.8  c.c.  of  NaOH,  or  2f  hours  in  that  containing  0 . 4  c.c, 
developed  into  larvae.^ 

Hence  it  can  be  seen  that  just  as  in  the  case  of  the  eggs  of 
purpuratus  and  of  Polynoe,  the  hypertonic  solution  as  a  rule  only 
affects  the  eggs  of  Lottia  when  a  certain  degree  of  concentration 
of  hydroxylions  (about  10"^  N)  has  been  reached.  Further,  it 
is  also  clear  that  when  the  concentration  of  hydroxylions  in  the 
hypertonic  solution  is  higher  than  in  sea-water,  the  number  of 
larvae  formed  also  increases.  But  in  this  case  also,  as  in  the 
experiments  with  the  eggs  of  S.  purpuratus,  the  optimal  con- 
centration of  hydroxylions  for  the  production  of  larvae  is  sub- 
ject to  large  variations  with  the  eggs  of  different  females. 

The  second  fact  brought  out  by  these  experiments  is  the 
importance  of  oxygen.  In  flat  dishes  (that  are  covered  against 
evaporation  of  the  sea-water)  in  which  the  eggs  lie  close  to  the 
surface  of  the  water,  the  number  of  eggs  that  develop  is  appreci- 
ably higher  than  in  dishes  in  which  the  eggs  are  covered  with  a 
deep  layer  of  sea-water. 

Finally,  it  is  a  point  of  interest  that  no  visible  membrane 
formation  takes  place  in  these  forms.  In  this  respect  the  eggs 
behave  differently  from  those  hitherto  discussed. 

Attempts  to  separate  by  an  interval  of  time  the  effect  of  the 
alkali  and  the  raising  of  the  osmotic  pressure,  as  can  be  done 
with  the  eggs  of  the  sea-urchin  and  of  Polynoe,  have  not  yet 
proved  successful  with  Lottia,  Moreover,  development  is  not 
produced  by  treatment  with  alkali  alone. 

In  all  these  experiments  the  eggs  developed  practically 

1  Loeb,  "Ueber  die  ailgemeiaen  Methoden  der  kunstlichen  Parthenogenese," 
op.  cit. 


Experiments  with  the  Eggs  of  Molluscs         269 

without  segmentation.  Kostanecki^  found  this  in  his  experi- 
ments with  Mactra  and  the  writer  confirmed  this  for  Lottia. 
It  must  be  remembered,  however,  that  these  are  onl}-  short- 
comings of  the  method,  and  it  was  therefore  to  be  expected  that 
different  methods  would  give  different  results. 

The  writer  did  not  succeed  in  causing  artificial  partheno- 
genesis in  the  eggs  of  Cumingia,  a  mollusc  at  Woods  Hole,  by 
any  of  the  older  methods,  but  Wasteneys  and  he  succeeded  in 
not  only  causing  these  eggs  to  develop,  but  also  to  develop  with 
normal  segmentation  by  treating  them  with  ox  blood.  The 
method  used  was  as  follows.  The  eggs  were  sensitized  to  the 
effects  of  serum  by  placing  them  for  from  two  to  four  minutes 
into  a  3/8  m  solution  of  strontium  chloride.  They  were  then 
placed  for  five  minutes  in  ox  serum  rendered  isotonic  with  sea- 
water  and  diluted  with  an  equal  part  of  a  m/2  solution  of 
NaCl+CaCls+KCl.  After  having  been  freed  from  all  traces 
of  serum  by  repeated  washing  in  a  Ringer  solution,  they  are 
transferred  for  sixty  minutes  into  hypertonic  sea-water  (50  c.c. 
sea-water +8  c.c.  2|  m  NaCl).  Control  experiments  showed 
that  the  treatment  with  serum  is  an  essential  factor  in  this 
process.^ 

This  latter  experiment  shows  the  close  similarity  of  the 
methods  of  artificial  parthenogenesis  in  various  groups  of 
animals. 

1  Kostanecki,  "Zur  Morphologie  der  kiinstlich  parthenogenetischen  Ent- 
wicklung  bei  Mactra,"  Arch.  f.  mikroskop.  Anat.  u.  Entwicklungsgesch.,  LXXII, 
327,  1908. 

2  Loeb  and  Wasteneys,  "  Fertilization  of  the  Eggs  of  Various  Invertebrates  by 
Ox  Serum,"  Science.  XXXVI,  255.  1912. 


XXVIII 

ARTIFICIAL  PARTHENOGENESIS  IN  THE  EGGS  OF 

FROGS 

During  the  first  years  of  his  work  and  later  the  writer 
vainly  appUed  the  chemical  methods  of  artificial  parthenogenesis 
to  the  eggs  of  fishes  and  of  frogs.  The  reason  for  this  failure 
may  possibly  lie  in  the  relative  impermeability  of  the  walls  of 
these  eggs  for  the  chemicals  used.  The  walls  of  the  eggs  of 
Fundulus  are,  as  long  as  they  are  normal,  not  only  impermeable 
for  salts  but  also  for  water.  It  seemed  desirable  that  a  method 
of  artificial  parthenogenesis  for  vertebrates  should  be  found 
since  it  is  so  much  easier  to  raise  the  larvae  of  vertebrates  than 
of  invertebrates.  It  is  under  the  circumstances  no  surprise  that 
such  a  method  was  found  almost  accidentally.  In  1907  Guyer 
pubUshed  a  paper  in  which  he  reported  that  by  injecting  lymph 
or  blood  into  the  unfertilized  eggs  of  frogs  he  succeeded  in 
starting  development  and  even  in  obtaining  two  tadpoles. 
Considering  the  importance  of  these  experiments  and  since 
they  seem  to  have  been  overlooked,  the  writer  feels  justified 
in  quoting  part  of  Guyer's  note: 

During  three  successive  springs  (1905-7)  the  writer  has  experi- 
mented on  unfertiUzed  frog  eggs  by  injecting  them  with  blood  or 
lymph  of  either  male  or  female  frogs.  In  all  some  fifteen  Imn.lred 
eggs  have  been  so  operated  upon.  Shortly  before  the  time  for  laying, 
the  eggs  were  taken  from  the  uterus  with  every  precaution  to  prevent 
contamination  by  sperm.  Those  nearest  the  cloacal  opening  were 
always  set  aside  as  a  control  and  in  not  a  single  instance  did  any  of  thorn 
develop.  The  other  eggs  were  pricked  with  a  very  fine-pointed  capil- 
lary tube  which  had  previously  been  charged  with  lymph  and  cor- 
puscles by  dipping  it  into  the  lymph  or  the  l)lood  of  another  frog. 

In  eggs  so  treated  numerous  instances  of  cell  proliferation  and 
embryon'ic  development  have  been  observed,  provided  the  eggs  were 


271 


272     Artificial  Parthenogenesis  and  Fertilization 


fully  matured  and  ready  for  fertilization.  Many  eggs  after  six  or 
eight  days  showed  upon  sectioning  that  they  had  approximated  the 
full  blastular  and  in  some  cases  the  gastrular  stages,  although  the 
condition  came  about  apparently  by  some  sort  of  internal  nuclear 
arrangement,  as  no  superficial  cleavage  furrows  were  observable  and 
no  demarkation  into  cells  was  visible  from  the  exterior  until  the  third 
or  fourth  da}-,  when  close  inspection  showed  in  some  cases  numerous 
small  vesicular  or  cellular  outlines. 

In  some  instances  definite  organs  were  developed,  though  fre- 
quently distorted  and  misplaced.  Cross-sections  of  one  embryo,  for 
example,  showed  such  pronounced  defects  as  two  neural  tubes  an- 
teriorly. Of  the  whole  number  of  eggs  operated  upon  only  two  devel- 
oped into  free-swimming  tadpoles  and  these  were  apparently  normal 
as  far  as  superficial  examination  disclosed.  They  have  not  yet  been 
sectioned.  After  sixteen  dsivs  one  died  and  the  other  was  killed  to 
insure  proper  fixation  for  histological  study. 

Apparently  the  white  rather  than  the  red  corpuscles  are  the  stimu-^ 
lating  agents  which  bring  about  development,  because  injections  of 
lymph,  which  contains  only  white  corpuscles,  produce  the  same  effect 
as  injections  of  blood.  Whether  or  not  the  fluid  part  of  the  lymph  or 
blood  produced  any  effect  could  not  be  definitely  determined  from  the 
material  at  hand.^ 

Guyer  thought  that  probably  the  cells  which  he  intro- 
duced were  developing  and  not  the  egg.  He  did  not  recognize 
that  his  experiment  was  a  case  of  artificial  parthenogenesis. 
This,  however,  does  not  detract  from  the  fact  that  he  was 
the  first  to  cause  the  development  of  the  unfertilized  egg  of 
the  frog  by  puncturing  it,  that  he  introduced  blood  into  the 
egg  for  this  purpose,  and  that  he  succeeded  in  producing  two 
parthenogenetic  tadpoles. 

Guyer's  results  were  to  a  large  extent  confirmed  by  Batail- 
lon.  Bataillon  found  that  mere  puncturing  of  the  egg  of  the 
frog  by  a  very  fine  needle  could  not  produce  any  embryogenesis 
but  that  a  second  factor  was  necessary,  namely,  that  some  of  the 
body  liquids  (blood  of  the  frog  or  newt  or  fish)  have  to  enter  the 
egg.     "A  considerable  percentage  of  the  eggs  of  Rana,  touched 

1  Guyer.  Science,  XXV,  910.  1907. 


Artificial  Parthenogenesis  in  Frogs  273 


with  the  blood  and  immediately  punctured,  will  develop,  while 
the  same  eggs  squeezed  out  from  the  female  frog  will  not  develo]) 
(when  punctured)."  Bataillon  states  that  in  the  eggs  thus 
treated  ''he  found  at  the  beginning  of  the  divisions  outside  the 
kinetic  figures,  chromatin  fragments  accompanied  by  asters, 
which  fragments  come  probably  from  elements  inoculated  with 
the  needle."^  In  a  recent  paper  Bataillon  reaches  the  conclu- 
sion that  it  is  the  leucocytes  which  cause  the  development  (1913). 

A  number  of  authors  have  succeeded  in  repeating  these 
experiments  by  Guyer  and  Bataillon,  Dehorne,^  Henneguy,^ 
Brachet,'*  McClendon,^  and  Loeb  and  Bancroft.^  The  last 
named,  however,  have  produced  tadpoles  also  without  the  use 
of  blood  or  lymph. 

While  the  number  of  eggs  which  begin  to  segment  when 
punctured  is  not  inconsiderable,  very  few  reach  the  tadpole 
stage.  There  is  a  difference  in  the  response  of  the  eggs  of 
various  kinds  of  frogs  to  this  treatment. 

The  number  of  unfertilized  eggs  which  began  to  segment 
after  puncture  was  according  to  Loeb  and  Bancroft  greater  in 
the  wood  frog  than  in  the  leopard  frog,  and  amounted  in  the 
most  favorable  cases  to  about  40  per  cent  in  the  former.  Only 
2  of  about  10,000  punctured  eggs  of  the  wood  frog  reached  the 
tadpole  stage,  but  these  died  before  they  were  able  to  swim. 
The  percentage  of  eggs  of  the  leopard  frog  which  reached  the 
tadpole  stage  was  greater.  From  700  punctured  eggs  of  the 
Southern  leopard  frog,  13  good  morulae  were  isolated  the  next 
day.     On  the  third  da}',  when  the  fertilized  controls  were  in  the 

1  Bataillon,  "La  parthenogenese  experimentale  des  amphibiens,"  Revue 
generale  des  Sciences,  XXII,  786,  1911;  Compt.  rend.  Acad.  Sc,  CL,  996,  1910; 
CLII,  920,  1911;  CLII,  1120.  1911;  CLII.  1271,  1911;  CLVI,  812,  1913;  Arch, 
de  Zool.  expcr.  el  gen.,  XLVI,  103,  1910. 

2  Dehorne,  Compt.  rend.  Acad.  Sc,  CL,  1-451,  1910. 

3  Henneguy,  Compt.  rend.  Acad.  Sc,  CLII,  941,  1911. 

*  Brachet,  Arch,  de  Biol.,  XXVI,  337,  1911. 

5  McClendon,  Am.  Jour.  Physiol.,  XXIX,  298,  1911. 

*  Loeb  and  Bancroft,  Jour.  Exper.  Zool.,  XIV,  275,  1913. 


274     Artificial  Parthenogenesis  and  Fertilization 


gastrula  stage,  13  unfertilized  punctured  eggs  were  also  in  the 
gastrula  stage  and  4  more  eggs  were  developing  abnormally. 
On  the  fourth  day,  8  of  the  parthenogenetic  eggs  had  good 
medullary  folds  and  4  had  irregular  folds.  On  the  sixth  day 
most  of  the  fertilized  eggs  hatched  and  8  of  the  parthenogenetic 
eggs  hatched  also.  Of  these  latter,  4  were  developing  regularly 
and  4  irregularly.     Those  that  had  not  hatched  were  abnormal. 

On  the  eighth  day,  the  larvae  arising  from  the  fertilized  eggs 
were  swimming.  Among  the  larvae  arising  from  the  unferti- 
lized punctured  eggs  only  3  were  normal,  and  their  develop- 
ment was  slightly  retarded,  perhaps  one  day.  In  addition,  6 
parthenogenetic  larvae  were  abnormal  but  still  alive. 

On  the  thirteenth  day,  2  of  the  parthenogenetic  larvae  were 
feeding  and  these  were  the  only  ones  which  survived  definitely. 
The  other  parthenogenetic  larvae  all  died  during  the  next  few 
days.  Of  the  2  surviving  larvae,  one  went  through  meta- 
morphosis after  five  months.  When  it  died,  the  tail  was  almost 
completely  absorbed  (Fig.  76).  Its  death  was  probably  acci- 
dental. The  other  lived  (Fig.  77)  a  month  longer  and  formed 
small  hind  legs,  but  died  in  the  tadpole  stage.  A  repetition  of 
these  experiments  by  the  same  authors  showed  that  only  the 
eggs  of  Rana  sphenocephala  and  R.  pipiens  produce  tadpoles 
when  punctured,  while  the  unfertilized  eggs  of  R.  silvatica, 
Chorophilus  feriarum,  and  of  Bufo  americanus  only  begin  to 
segment   but   die   before   the   tadpole   stage   when   they    are 

punctured. 

As  far  as  the  effect  of  puncturing  the  egg  is  concerned,  it 
may  be  comparable  to  the  effect  of  agitation  upon  the  starfish 
egg.  The  latter  forms  a  membrane  as  an  effect  of  agitation 
and  it  may  be  that  the  destruction  of  the  surface  layer  of  the 
frog  egg  is  the  essential  feature  in  this  "traumatic"  partheno- 
genesis. While  the  spermatozoon  causes  the  destruction  of  the 
cortical  layer  of  an  egg  by  a  chemical  substance  (a  "lysin"); 
and  while  the  same  can  be  accomplished  in  the  egg  of  some 


Artificial  Parthenogenesis  in  Frogs  275 

starfish  and  some  annelids  by  gentle  agitation,  in  the  egg  of 
the  frog  it  can  be  done  by  puncturing  the  surface  layer. 
Future  experiments  will  decide  whether  or  not  the  leucocytes 
play  the  role  of  the  second  factor  which  Bataillon  ascribes  to 
them. 

Loeb  and  Bancroft  tried  to  ascertain  the  sex  of  the  par- 
thenogenetic    frog    and   tadpole   which    they    obtained.     The 


Fig.  76  Fig.  77 

Figs.  76  and  77. — Parthenogenetic  frog  and  parthcnogenetic  tadpole  (nat- 
ural size). 

gonads  contained  eggs  in  both  cases.  This  at  first  sight  might 
be  taken  to  indicate  that  both  were  females,  but  the  problem  is 
complicated  by  the  fact  found  by  Pfliiger,  and  recently  worked 
out  by  Kuschakewitsch,^  that  very  often  in  the  early  stages  the 
gonads  of  a  frog  may  contain  eggs  which  afterward  degenerate. 
Such  ''intermediates"  may  develop  into  males. 

The  gonads  of  the  parthenogenetic  frog  obtained  by  Loeb 
and  Bancroft  agreed  with  Kuschakewitsch's  description  of 
an  "intermediate"  which  is  in  the  process  of  developing  into  a 
male. 

1  Kuschakewitsch,  Festschrift  f.  Richard  Hertwig,  Bd.  II.  p.  145,  I'JIO. 


XXIX 

ARTIFICIAL  PARTHENOGENESIS  IN  PLANTS 

Experiments  on  artificial  parthenogenesis  in  plants  are 
limited  by  the  fact  that  it  is  necessary  for  this  purpose  to  obtain 
the  unfertilized  eggs  in  large  numbers  and  that  the  eggs  are  not 
naturally  parthenogenetic.  The  Fucaceae  seem  to  meet  this 
requirement  and  J.  B.  Overton,  of  the  University  of  Wisconsin, 
has  recently  succeeded  in  bringing  about  artificial  partheno- 
genesis in  a  species  of  Fucus  at  Woods  Hole.  Since  only  a  pre- 
liminary report  has  thus  far  appeared^  on  this  subject  it  may  be 
best  to  quote  from  it. 

Fucus  vesiculosus,  a  dioecious  species,  occurs  near  the  shores 
between  tide  marks  at  Woods  Hole,  and  plants,  both  in  the  vegeta- 
tive and  reproductive  conditions,  are  usually  abundant.  The  sperma- 
tozoids  and  oospheres  are  usually  discharged  sparingly  during  ebb 
tide  and  abundantly  during  flood  tide.  Plants  were  collected  during 
ebb  tide,  the  distal  portions  removed  and  placed  in  dishes  on  ice  over 
night.  Care  was  taken  that  the  conceptacles  bearing  eggs  and  sperms 
were  kept  separate.  When  it  was  desired  to  obtain  the  eggs  and 
sperms,  dishes  containing  conceptacles  were  filled  with  fresh  sea-water, 
or  after  first  exposing  the  conceptacles  to  a  hypotonic  sea-water,  when 
eggs  and  sperms  were  discharged  in  large  numbers.  The  freshly  ex- 
truded eggs  drop  to  the  bottom  of  the  dishes  and  can  be  taken  up  with 
a  pipette  and  transferred  to  watch  glasses  for  experiment.  After  a 
short  time  the  eggs  show  a  tendency  to  adhere  very  firmly  to  the 
bottom  of  the  watch  glasses  so  that  fluids  can  easily  be  poured  off 
and  others  added  without  losing  the  eggs. 

In  plants  used  to  induce  cell  division  by  artificial  means  great 
care  was  taken  to  prevent  contamination  by  sperm.  The  female 
plants  were  carefully  washed  whh  fresh  water  to  kill  any  sperms  wliich 
might  adhere  to  them.  None  of  the  eggs  obtained  from  such  sterilized 
plants  ever  developed  in  the  numerous  controls,  which  were  run  in 

ij.  B.  Overton,  "Artificial  Parthenogenesis  in  Fucus,"  Science,  XXXVII, 
841,  1913. 

277 


278    Artificial  Parthenogenesis  and  Fertilization 

connection  with  the  experiments,  showing  beyond  a  doubt  that  the 
female  plants  treated  were  absolute^  sterile. 

Loeb  has  shown  that,  when  unfertilized  eggs  of  the  sea-urchin  are 
placed  for  one  and  one-half  to  two  minutes  in  a  mixture  of  50  c.c.  of 
sea-water+3  c.c.  of  0. 1  m  acetic,  butyric,  or  other  fatty  acid  and  then 
transferred  to  normal  sea-water,  a  fertilization  membrane  is  formed. 
This  method  was  applied  to  unfertilized  Fiicits  eggs.  In  experimenting 
with  the  eggs  those  used  at  any  one  time  were  always  divided  into  three 
lots.  One  lot  was  used  as  a  control,  another  was  fertilized,  and  the 
third  was  treated  with  the  solution.  If  a  single  egg  in  the  control 
formed  a  cell-wall,  which  seldom  happened,  the  three  lots  were  dis- 
carded. In  case  the  eggs  were  treated  with  acetic  or  butyric  acid^ 
as  above  described,  a  large  number  of  them  formed  in  about  ten 
minutes  a  membrane  or  cell-wall  which  was  exactly  similar  to  the  one 
formed  about  normally  fertilized  eggs.  By  plasmolyzing  the  eggs  the 
membrane  is  readily  seen.  Eggs  not  treated  with  a  solution  or  not 
fertilized  undergo  C3i;olysis  and  degenerate.  In  any  case  many  of  the 
eggs  failed  to  develop,  but  about  one-fourth  as  many  formed  mem- 
branes under  the  influence  of  the  solutions  as  were  formed  about  ferti- 
hzed  eggs.  After  the  formation  of  the  membranes  if  the  eggs  are 
placed  in  hypertonic  sea-water,  8  c.c.  to  10  c.c.  of  2.5  m  NaCl  or  KCl 
+50  c.c.  sea-water,  for  30  minutes  and  are  then  brought  back  into 
normal  sea-water,  development  continues.  Nearly  all  of  the  eggs 
which  have  formed  a  membrane  become  pear-shaped,  showing  a 
rhizoidal  papilla,  and  by  next  morning  have  cleaved.  The  rhizoidal 
cell  is  cut  off  and  one  or  more  cleavages  have  taken  place  in  the  other 
portion  of  the  sporeling.  If  the  cultures  are  properly  aerated,  spore- 
lings  develop  resembling  in  every  respect  those  grown  from  fertilized 
eggs.  In  place  of  sea- water  containing  a  fatty  acid,  solutions  of 
various  other  cytolytic  substances  were  used,  but  none  stimulated 
membrane  formation  or  development  as  well  as  the  acids. 

With  regard  to  the  first  formation  of  the  cell-wall  over  the  surface 
of  isolated  masses  of  plant  protoplasm,  it  is  usually  attributed  to  a 
process  of  secretion  by  the  outer  layer.  That  the  process  is  a  rapid 
one  is  shown  by  the  fact  that  in  Fucus  eggs  a  cell-wall  is  formed  in  ten 
minutes  after  the  entrance  of  the  sperm.  Cell-wall  formation  may  also 
be  artificially  induced,  as  shown  above,  by  various  substances.  In 
some  cases  a  cell-wall  may  appear  under  certain  conditions  on  the  sur- 
face of  plasmolyzed  protoplasts  in  fifteen  minutes,  as  has  been  shown 
by  Klebs,  Palla,  and  others,  while  in  other  cases  hours  are  required 


Artificial  Parthenogenesis  in  Plants  271) 


for  wall  formation.  It  would  appear  that  the  action  of  the  acids  in 
inducing  a  cell-wall  to  be  formed  about  the  unfertilized  Fucm  eggs  is 
similar  to  the  action  which  calls  forth  membrane  formation  in  the 
animal  egg. 

The  fact  that  the  method  which  causes  artificial  partheno- 
genesis in  the  eggs  of  many  animals  acts  in  the  same  way 
in  the  case  of  the  eggs  of  plants  indicates  the  identity  of  this 
process  in  all  living  organisms. 


XXX 

PRESERVATION  OF  THE  LIFE  OF  THE  EGG  BY  THE  ACT 

OF  FERTILIZATION 

1.  The  unfertilized  egg  dies  in  a  comparatively  short  time, 
while  the  act  of  fertilization  saves  the  life  of  the  egg  and  allows 
it  to  give  rise,  theoretically  at  least,  to  an  unlimited  series  of 
generations.  The  question  arises :  How  does  the  spermatozoon 
save  the  life  of  the  egg  ?  It  is  not  necessary  to  emphasize  the 
physiological  importance  of  this  feature  of  the  process  of  ferti- 
lization. 

The  rapidity  with  which  the  unfertilized  eggs  die  differs 
according  to  the  species.  The  unfertilized  egg  of  the  starfish 
(Asterias)  dies  at  summer  temperature  in  a  few  hours,  that  of 
Polynoe  in  less  than  a  day,  while  that  of  the  sea-urchin  (Arbacia) 
may  live  for  a  week  or  longer.  In  all  cases  the  life  of  the 
unfertilized  egg  can  be  prolonged  if  its  oxidations  are  suppressed  ; 
and  the  rapidity  with  which  they  die  seems  to  depend  upon  the 
relative  velocity  of  oxidations  in  the  unfertilized  egg. 

As  already  mentioned,  the  eggs  of  the  starfish  are  mostly 
unripe  when  laid,  and  they  only  begin  to  ripen  in  sea-water. 
The  different  eggs,  however,  do  not  all  ripen  at  the  same  rate. 
Now  I  found  that  if  mature  eggs  were  not  at  once  or  soon 
fertilized  or  caused  to  develop  by  chemical  means,  they  quickly 
disintegrate,  i.e.,  in  the  course  of  a  few  hours. 

The  cytoplasm  of  the  living  eggs  of  Asterias  is  homogeneous 
and  of  a  light  yellowish  color.  This  appearance  the  eggs  also 
retain  after  maturation,  as  long  as  they  are  alive;  nor  do  they 
lose  it  if  development  is  initiated  by  the  entry  of  a  spermatozoon 
or  by  the  methods  of  artificial  parthenogenesis. 

If,  however,  the  ripe  eggs  remain  unfertilized  or  are  not 
caused  to  develop,  they  die  in  the  course  of  from  four  to  twelve 

281 


282     Artificial  Parthenogenesis  and  Fertilization 


hours,  and  this  process  is  accompanied  by  a  characteristic 
alteration  in  the  color  of  the  egg.  It  becomes  first  dark,  then 
rapidly  black,  and  instead  of  the  homogeneous  appearance  of 
the  protoplasm,  there  appear  large  drops  or  globules  within  it. 
If  we  examine  with  a  microscope  such  a  culture  of  unfertilized 
eggs  after  about  twenty-four  hours,  we  find  two  classes  of  eggs: 
first  the  ripe  but  uniformly  dark,  dead  eggs  and,  second,  the 
immature  but  living,  normally  colored  eggs.  We  stated  in  a 
previous  chapter  that  the  eggs  which  are  taken  from  the  ovary 
of  a  starfish  do  not  all  ripen  at  once ;  many  mature  very  slowly, 
others  practically  never.  It  can  now  readily  be  observed  that 
the  unripe  eggs  remain  alive  several  days  longer,  until  they  even- 
tually fall  a  prey  to  bacteria,  while  the  ripe  eggs  mostly  become 
dark  and  die  in  from  four  to  eight  hours  after  maturation. 

Is  the  death  of  the  ripe  egg,  which  has  not  been  made  to 
develop,  due  to  intrinsic  processes  or  to  the  bacteria  contained 
in  the  sea-water?  A  reliable  method  of  determining  this  is 
to  make  pure  sterile  cultures  of  the  eggs  in  sea-water.  This  is 
comparatively  simple  with  starfish  eggs.  Eight  flasks  were 
sterilized,  filled  with  sterilized  sea-water,  and  then  heated  again 
for  twenty  minutes  to  100°  on  three  consecutive  days.  A 
female  starfish  was  thoroughly  washed  externally,  one  arm 
opened,  and  an  ovary  removed  with  sterilized  forceps  and 
brought  into  sterilized  sea-water.  From  the  thick  stream  of 
eggs  which  immediately  flowed  out  of  the  ovary  a  couple  of 
drops  were  quickly  placed  in  each  of  the  sterilized  flasks  with  a 
sterile  pipette.  A  second  series  of  eight  flasks  contained  normal, 
unsterilized  sea-water  and  in  each  of  these  flasks  also  a  couple 
of  drops  of  the  same  eggs  were  placed.  A  third  series  of  flasks 
was  filled  with  sea-water,  to  which  2  c.c.  of  a  thoroughly  putrid 
culture  of  old  starfish  eggs  had  been  added  in  order  to  cause 
from  the  first  a  vigorous  development  of  bacteria.  These 
flasks  too  contained  eggs  from  the  same  female  as  the  sterilized 
flasks. 


Preservation  of  the  Life  of  the  Egg  283 

That  the  first  eight  flasks  had  been  thoroughly  steriHzed 
was  demonstrated  by  the  fact  that  all  the  flasks  remained  per- 
fectly clear  and  unclouded  throughout  tlie  whole  experiment; 
and  that  three  flasks  which  were  not  opened  till  the  end  of  the 
experiment,  i.e.,  after  ten  weeks,  were  quite  clear  and  each  single 
egg  therein  could  be  distinctly  recognized.  The  flasks  with 
unsterilized  sea-water  became  cloudy  after  only  twenty-four 
hours,  and  after  two  days  the  eggs  had  fallen  a  prey  to  bacteria 
and  no  egg  was  any  longer  recognizable.  The  sterilized  flasks 
which  were  opened  were  always  quite  free  from  odor,  while 
the  unsterilized  flasks  already  smelled  unendurably  putrid  after 
one  to  two  days.  A  microscopical  examination  of  the  sea-water 
for  bacteria  always  remained  absolutely  negative  in  the  steril- 
ized flasks,  and  was  always  quite  positive  in  the  other  flasks. 
In  the  flasks  to  which  2  c.c.  of  the  putrid  culture  of  starfish 
eggs  had  been  added,  bacteria  and  infusoria  were  extremely 
numerous  from  the  first. 

Six  hours  after  the  beginning  of  the  experiment,  one  flask 
of  each  of  the  three  series  was  opened,  and  the  eggs  examined 
with  a  microscope.  The  appearance  was  the  same  in  all  three 
flasks:  practically  all  the  eggs  were  ripe,  and  a  small  number 
were  dark  or  black.  What  is  of  definite  importance  for  us  is 
the  fact  that  the  percentage  of  dark,  dead  eggs  was  quite  as 
large  in  the  sterile  culture  as  in  the  unsterilized  or  befouled 
sea-water. 

Twelve  hours  later,  and  therefore  eighteen  hours  after  the 
experiment  was  started,  another  flask  from  each  of  the  three 
cultures  was  opened.  This  time  practically  all  the  eggs  in  the 
sterile  culture  were  dark  or  black,  and  a  few  already  showed 
decomposition  into  globules.  The  same  percentage  of  eggs 
in  the  two  other  cultures  was  also  dark.  Hence  the  eggs  die 
just  as  quickly  in  the  sterilized  flasks,  which  are  absolutely  bacteria- 
free,  as  in  the  flasks  which  contain  bacteria.  Death  takes  place 
from  intrinsic  causes  and  so  quickly  that  the  scanty  bacteria 


284    Artificial  Parthenogenesis  and  Fertilization 

present  in  the  sea-water  are  scarcely  able  to  accelerate  the 
death  of  the  eggs.  The  eggs  have  indeed  already  died  from 
intrinsic  factors  before  the  bacteria  can  enter. 

The  flasks  opened  later  only  confirmed  the  above.  Each 
one  opened  in  the  first  few  days  contained  also  a  small  number 
of  living  eggs  of  clear-cut  appearance.  The  latter  were,  how- 
ever, without  exception,  unripe.  The  experiment  therefore 
proved  that  the  ripe  eggs  of  the  starfish  decay  in  a  few  hours, 
and  the  causes  of  their  death  must  not  be  looked  for  in  the 
bacteria  of  the  sea-water;  and,  further,  that  under  the  same 
conditions  the  unripe  eggs  remain  alive. 

An  experiment  was  now  made  to  find  out  whether  the  life 
of  the  unripe  eggs  can  be  prolonged  by  preventing  them  from 
ripening.  As  previously  mentioned,  maturation  requires  the 
presence  of  free  oxygen.  Freshly  laid  eggs  of  Asterias  were 
divided  into  two  series  of  eight  flasks.  One  series  of  flasks  was 
connected  with  a  hydrogen  generator,  the  other  with  a  cylinder 
containing  pure  oxygen.  Before  the  beginning  of  the  experiment, 
all  the  air  in  the  one  series  of  flasks  was  driven  out  by  a  stream 
of  hydrogen,  and  a  vigorous  stream  of  hj^drogen  was  maintained 
throughout  the  experiment.  Both  series  of  flasks  contained 
freshly  gathered  unripe  eggs  of  Asterias.  The  experiment 
continued  for  three  days,  and  from  time  to  time  one  flask  was 
disconnected  and  its  contents  examined.  The  eggs  which  were 
exposed  to  the  stream  of  oxygen  matured  as  quickly  and  in  as 
large  numbers  as  in  normal  sea-water,  and  the  ripe  eggs  died 
as  soon.  In  the  stream  of  hydrogen,  maturation  did  not  take 
place  in  the  majority  of  the  eggs  and  they  remained  alive.  A 
vigorous  development  of  bacteria  took  place  in  the  hydrogen 
culture  while  there  was  none  or  only  a  weak  one  in  the  oxygen 
culture. 

The  death  and  disintegration  of  the  eggs  is  also  prevented 
by  treatment  with  acid,  which,  as  previously  mentioned,  pre- 
vents the  maturation  of  the  eggs  (without  killing  them) .     Eggs 


Preservation  of  the  Life  of  the  Egg 


285 


which  are  placed  for  ten  or  fifteen  minutes  in  100  c.c.  of  sea- 
water+4c.c.  N/10  HCl,  without  coming  into  contact  with 
alkaline  sea-water,  ripen  much  more  slowly,  generally  not  at 
all,  when  after  that  time  they  are  replaced  in  normal  sea-water. 
They  retain  also,  as  long  as  they  are  unripe,  the  full  normal 
appearance  of  living  eggs,  until  they  eventually  fall  victims 
to  bacteria.  Unripe  eggs  too,  when  placed  in  neutral  sea-water, 
do  not  ripen  for  the  most  part,  and  retain  their  normal  appear- 
ance if  they  remain  immature. 

It  appears  to  follow  from  this  experiment  that  the  same 
process  which  lies  at  the  bottom  of  the  maturation  of  the  star- 
fish egg  also  leads  to  its  death,  unless  it  is  prevented  by  those 
measures  which  we  designate  as  fertilization.  I  now  tried 
whether  it  is  possible  to  keep  the  ripe  eggs  alive  longer  through 
lack  of  oxygen.  In  fact  I  obtained  some  positive  results  in 
this  connection.  Starfish  eggs  were  spread  out  in  a  thin  layer: 
After  three  hours  75  per  cent  of  the  eggs  had  ripened.  A 
part  of  the  ripe  eggs  was  immediately  placed  in  narrow 
glass  tubes,  in  which  the  deeper  layers  suffer  the  same  lack 
of  oxygen.  A  second  portion  was  placed  in  small  flasks, 
through  which  was  led  a  constant  stream  of  pure  ox>'gen. 
Next  morning,  fifteen  hours  after  the  eggs  were  placed  in  the 
oxygen,  the  different  portions  of  eggs  were  tested.  Those  eggs 
placed  in  the  current  of  oxygen  showed  in  one  dish  98  per  cent 
of  ripe,  dark,  and  dead  eggs,  and  2  per  cent  of  unripe,  living 
eggs.  The  eggs  remaining  in  normal  sea-water  contained  as 
before  some  78  per  cent  of  ripe  eggs,  which,  however,  were  all 
black  and  dead,  with  the  exception  of  a  few  which  had  begun  to 
divide  and  were  alive.  The  unripe  eggs  were  also  living.  On 
the  other  hand,  those  eggs  which  had  been  in  the  glass  tubes,  in 
complete,  or  relatively  complete,  lack  of  oxj^gen,  were  nearly 
all  alive.  This  observation,  in  fact,  indicates  that  the  same 
process  which  leads  to  the  maturation  of  the  egg  also  causes  the 
death  of  the  egg  unless  it  is  prevented  in  time.     In  this  way 


286     Artificial  Parthenogenesis  and  Fertilization 

fertilization  becomes  an  act  which  saves  or  prolongs  life.^ 
A  simpler  way  of  prolonging  the  duration  of  life  of  the  mature 
but  unfertilized  egg  of  the  starfish  consists  in  adding  a  trace  of 
KCN  to  the  sea-water  (about  5  or  6  drops  of  1/10  of  1  per  cent 
KCN  to  50  c:c.  of  sea-water). 

A.  P.  Mathews  has  continued  these  experiments  and  like- 
wise found  that  the  life  of  the  unfertilized  ripe  eggs  can  be  pro- 
longed by  lack  of  oxygen.^ 

This  proves  that  the  death  of  the  mature  but  unfertilized 
egg  is  if  not  determined  at  least  accelerated  by  oxidations. 

What  is  the  explanation  of  these  facts  ?  We  know  that  the 
unfertilized  but  mature  egg  of  the  starfish  is  the  seat  of  compara- 
tivel}'  rapid  oxidations  which  are  possibly  or  probablj'  lacking 
in  the  unripe  egg.  These  oxidations  lead  directly  or  indirectly 
to  the  death  of  the  mature  egg,  while  their  prevention  saves  the 
life  of  the  egg,  at  least  for  some  time.  The  unfertilized  mature 
egg  may  be  compared  to  an  anaerobe.  The  nature  of  the  life- 
saving  action  of  the  act  of  fertilization  may  then  be  expressed 
by  the  statement  that  by  the  act  of  fertilization  the  egg  is  trans- 
formed from  an  anaerobe  into  an  aerobe.  It  is  possible  that 
the  oxidations  do  not  kill  the  unfertilized  egg  directly,  but 
only  through  the  medium  of  physical  or  morphological  changes 
which  follow  or  accompany  the  oxidations. 

The  idea  that  fertilization  saves  the  life  of  the  egg  by  render- 
ing it  immune  against  oxidations  finds  support  -not  only  in  the 
fact  that  the  life  of  the  unfertilized  egg  of  the  starfish  is  pro- 
longed if  we  deprive  it  of  oxygen  or  inhibit  oxidations  through 
the  addition  of  KCN,  but  also  in  the  fact  that  the  unfertilized 
egg  of  the  sea-urchin  lives  much  longer  in  sea-water  than  does 
the  unfertilized  mature  egg  of  the  starfish.  This  should  lead 
us  to  expect  that  the  rate  of  oxidations  in  the  unfertilized  but 

1  Loeb,  "  Ueber  Eireifung,  naturlichen  Tod  und  Verlangerung  des  Lebens  beim 
unbefruchteten  Seesternei  (Asterias  forbesii)  und  deren  Bedeutung  fur  die  Theorie 
derBefruchtung,"  Pfluger's  Archiv,  XCIII,  59,  1902;    U ntersuchungen,  p.  237. 

2  A.  P.  Mathews,  Am.  Jour.  Physiol.,  XVIII,  89,  1907. 


Preservation  of  the  Life  of  the  Egg  287 


ripe  starfish  egg  is  comparatively  greater  than  in  the  sea-urchin 
egg.  This  is  the  case  since  the  unfertihzed  egg  of  the  sea-urchin 
has  a  rate  of  oxidations  from  four  to  six  times  smaller  than  that 
found  after  fertilization;  while  the  ripe  but  unfertilized  egg  of 
the  starfish  has  a  rate  of  oxidations  equal  to  that  of  the  fertilized 
egg^  (chap.  iii). 

We  have  another  fact  in  support  of  our  view.  We  saw  in  a 
previous  chapter  that  the  artificial  membrane  formation  raises 
the  rate  of  oxidations  in  the  sea-urchin  egg  about  six  times. 
If  our  contention  that  the  unfertilized  egg  is  comparable  to  an 
anaerobe  is  correct  the  membrane  formation  should  hasten  the 
death  of  the  unfertilized  sea-urchin  egg.  This  is,  as  we  saw, 
actually  the  case.  While  the  unfertilized  egg  of  purpuratus 
without  membrane  lives  for  several  days,  the  unfertilized  egg 
after  membrane  formation  dies  at  the  same  temperature  in  a 
few  hours.  Moreover,  we  saw  that  the  death  of  the  unferti- 
lized egg  after  membrane  formation  is  retarded  if  we  inhibit 
the  oxidations  in  the  egg.  We  pointed  out,  however,  that  in 
this  case  the  oxidations  may  lead  only  indirectly  to  the  rapid 
death  of  the  egg,  inasmuch  as  they  set  the  apparatus  of  nuclear 
cell  division  in  motion.  We  can  also  save  the  life  of  the  egg 
after  membrane  formation  by  inhibiting  development  with 
chloral  hydrate,  which  does  not  diminish  the  rate  of  oxida- 
tions. ^ 

2.  It  has  been  one  of  the  important  results  of  our  work  to 
show  that  for  the  egg  of  the  sea-urchin,  and  perhaps  in  general, 
the  causation  of  normal  development  requires  as  a  rule  the 
co-operation  of  two  factors,  the  membrane-forming  factor  and 
the  corrective  factor.  The  question  arises,  which  of  the  two 
has  the  life-saving  effect?  At  first  sight  it  would  seem  as  if 
this  important  function  was  to  be  attributed  to  the  second 
factor;    for  it  is  the  second  factor  which  saves  the  egg,  after 

1  Loeb  and  Wasteneys,  Archiv  f.  Entwicklungsmechanik,  XXXV,  555.  1912. 
^  Loeb  and  AVasdeneys,  Jour.  Biol.  Chem.,  XIV,  517,  1013. 


288     Artificial  Parthenogenesis  and  Fertilization 

membrane  formation,  from  the  threatening  disintegration.  Yet 
this  conclusion  would  be  wrong. 

The  unfertilized  mature  egg  of  S.  purpuratus  (in  which  no 
membrane  formation  has  been  called  forth)  lives  at  room  tem- 
perature about  two  or  three  days  and  then  disintegrates.  We 
saw  in  a  previous  chapter  that  the  application  of  the  second 
factor  (the  treatment  of  the  egg  with  the  hypertonic  solution) 
may  precede  the  artificial  membrane  formation  by  one  or  more 
days.  If  this  second  factor  alone  possessed  the  life-saving  action, 
we  should  expect  that  unfertilized  eggs  treated  with  the  hyper- 
tonic solution  alone  w^ould  live  longer  than  the  unfertilized  eggs 
not  treated  at  all.  This  is,  however,  not  the  case.  The  unferti- 
lized eggs  of  S.  purpuratus  treated  with  the  hypertonic  solution 
alone  die  as  quickly  as  the  non-treated  eggs.  The  same  eggs, 
however,  will  live  and  develop  if  the  membrane  formation  is 
called  forth.  In  this  case  we  might  say  that  the  membrane 
formation  has  the  life-saving  effect.  In  reality  it  is  the  com- 
bined action  of  both  by  which  the  Hfe  of  the  egg  is  prolonged. 

These  facts  also  make  it  plain  why  the  act  of  membrane 
formation  alone  can  save  the  life  of  certain  starfish  eggs  or 
the  eggs  of  certain  annelids  for  which  membrane  formation 
alone  suffices  to  induce  normal  development.  Not  all  the 
eggs  of  such  animals,  e.g.,  Asterina,  are  able  to  develop  through 
artificial  membrane  formation  alone,  but  only  a  small  number; 
and  we  have  reason  to  assume  that  in  these  eggs  the  second 
factor  is  formed  or  pre-exists  in  ample  quantity.  For  these 
eggs  the  mere  act  of  membrane  formation  is  sufficient  to  save 
their  lives,  since  they  are  supposed  to  supply  the  second  factor 
themselves. 

It  is  therefore  the  causation  of  development  and  not  the 
action  of  one  of  the  tw^o  factors  alone  which  saves  the  life  of 
the  unfertilized  egg. 

3.  We  have  mentioned  the  fact  that  lack  of  oxygen  or  the 
suppression  of  oxidations  prolongs  the  life  of  the  unfertilized 


Preservation  of  the  Life  of  the  Egg  289 


mature  egg,  while  fertilization,  which  makes  the  egg  immortal, 
raises  the  rate  of  oxidations  considerably,  at  least  in  the  egg 
of  the  sea-urchin.  We  have  seen  that  in  the  unfertilized  egg  of 
the  sea-urchin  oxidations  take  place  though  at  a  slow  rate. 
The  unfertilized  eggs  seem  to  perish  very  rapidly  througli 
these  oxidations.  We  have  stated  that  the  mature  unfertilized 
egg  resembles  an  anaerobe,  while  the  act  of  fertilization  trans- 
forms it  into  an  aerobe.  We  are  certainly  here  confronted 
with  one  of  the  most  interesting  and  far-reaching  features  of 
the  problem  of  fertilization.  The  unfertilized  egg  is  perhaps 
at  present  the  only  instance  of  a  cell  for  which  ''natural  death" 
can  be  proven.  The  act  of  fertihzation  or  rather  the  induce- 
ment of  development  procures  theoretical  immortality  for  the 
egg,  since  the  sex  cells  of  the  new  individual  to  which  the  egg 
gives  rise  are  parts  of  the  egg. 

The  oxidations  are,  however,  not  the  only  processes  which 
are  responsible  for  the  premature  ''natural  death"  of  the 
unfertilized  egg;  otherwise  it  should  be  possible  to  keep  it 
alive  indefinitely  without  oxygen,  which  is  not  the  case.  The 
act  of  fertilization  brings  about  a  profound  physicochemical 
modification  in  the  egg  which  is  not  confined  to  one  type  of 
chemical  reactions. 


XXXI 

ARTIFICIAL  PARTHENOGENESIS  AND  HEREDITY 

1.  The  spermatozoon  not  only  induces  the  development 
of  the  egg,  but  it  also  transmits  the  hereditary  characters  of 
the  male  parent  to  the  offspring.  The  possibility  of  artificial 
parthenogenesis  makes  it  certain  that  the  two  processes  are 
not  determined  by  the  same  substances,  since  the  methods  of 
artificial  parthenogenesis  are  analogous  for  very  diverse  species, 
while  the  hereditary  characters  are  very  different. 

When  closely  related  species  are  crossed  the  hereditary 
characters  of  the  male  are  of  course  recognizable  in  the  off- 
spring. The  question  arose:  What  would  happen  if  widely 
divergent  species  were  crossed,  such  as  sea-urchin  and  starfish  ? 
The  writer  found  a  method  of  bringing  about  this  hybridization. 
The  main  interest  was  whether  or  not  a  sea-urchin  egg  fertilized 
with  the  sperm  of  starfish  would  produce  the  skeleton  typical 
for  the  pluteus  stage  of  the  larva.  As  mentioned  above,  the 
hybridization  between  purpuratus  ?  and  Asterias  S  could  be 
accomplished  in  50  c.c.  sea-water +0 . 6  c.c.  N/10  NaOH.  These 
hybrid  eggs  segmented  at  the  same  rate  as  the  eggs  fertilized 
with  sperm  of  their  own  species,  and  the  development  was 
normal  up  to  the  gastrula  stage.  Then  the  eggs  began  to  die 
in  large  numbers  and  those  which  survived  were  sickly  and 
developed  at  a  much  lower  rate  than  the  eggs  fertilized  with 
purpuratus  sperm.  But  the  small  number  of  eggs  which  lived 
long  enough  developed  into  plutci  which  were  in  every  point 
identical  with  the  pure  breed  of  purpuratus.  In  the  case  of 
heterogeneous  hybridization  the  spermatozoon  produces  only 
the  developmental  but  not  the  hereditary  effect.^ 

I  Loeb,  "Ueber  die  Befruchtung  von  Seeigeleiern  (lurch  Seesternsanien," 
Pfiuger's  Archiv,  XCIX,  323,  1903;  "  Weitere  Versuche  ueber  heterogene  Hybri- 
disation bei  Echinodermen."  Pfiuger's  Archiv,  CIV.  32.5.  1904;  -'Heredity  in 
Heterogeneous  Hybrids."  Jour.  MorphoL,  XXIII.  1.  1912. 

291 


292     Artificial  Parthenogenesis  and  Fertilization 


This  statement  will  become  much  clearer  by  a  few  illustra- 
tions. Figs.  78-80  are  (five-days-old)  plutei  of  purpuratus 
produced   by   artificial   parthenogenesis.     They   are   identical 


Fig.  78 


Fie;.  79 


Fig.  80 


Figs.   78-80. 
parthenogenesis. 


-Five-days-old  plutei  of  S.  purpuratus,  produced  by  artificial 


with  the  pure  breed  of  purpuratus.  Figs.  81-83  are  three  plutei 
produced  by  fertilizing  the  sea-urchin  egg  (purpuratus)  with 
the  sperm  of  the  starfish.  Thej^  are  identical  with  the  plutei 
produced  by  artificial  parthenogenesis. 


Fig.  81  Fig.  82  Fig.  83 

Figs.  81-83. — Five-days-old  plutei  from  S.  purpuratus  ?  and  Asterias  S. 
Tlie  plutei  are  identical  with  the  parthenogenetic  plutei. 

If,  however,  the  egg  of  *S.  purpuratus  is  fertilized  with  the 
sperm  of  >S.  frandscanus,  a  closely  related  form,  a  pluteus  of 
an  altogether  different  type  is  produced  (Figs.  84-86).  The 
skeleton  has  the  rough  appearance  of  the  frandscanus  pluteus 
and  the  typical  arm  formation  characteristic  of  this  paternal 
form.  It  is  therefore  obvious  that  heterogeneous  hybridiza- 
tion is  in  reality  artificial  parthenogenesis  with  this  difference 


Artificial  Parthenogenesis  and  Heredity 


293 


only,  that  a  living  spermatozoon  carries  the  parthenogenetic 
substances  into  the  egg.  The  character  of  these  plutei  is 
always  the  same;  they  do  not  show  any  such  variations  as 
Tennent  reports. 

In  addition  to  fertilizing  the  eggs  of  *S.  purpuratus  with  the 
sperm  of  starfish,  the  writer  succeeded  also  in  fertilizing  the 
eggs  of  purpuratus  with  the  sperm  of  ophiurids  and  holothurians. 
Godlewski  used  the  same  method  with  success  for  the  fertiliza- 
tion of  the  egg  of  the  sea-urchin  with  the  sperm  of  a  crinoid 
(Antedon).    In  all  these  cases  the  larvae  were  purely  maternal.^ 


Fig.  85 


Fig.  86 


Fig.  84 

Figs.  84-86. — Five-days-old  plutei  of  S.  purpuratus  ?  and  S.  franciscanus  S. 
The  plutei  are  different  from  the  parthenogenetic  plutei. 

Godlewski  found  that  the  nuclei  of  crinoid  and  sea-urchin 
fuse.  The  chromosomes  of  Antedon  are  found  in  the  nuclei  of 
the  sea-urchin  egg  fertilized  with  Antedon  sperm.  The  eggs 
developed  normally  to  the  blast ula  stage,  when  a  retardation 
and  other  abnormalities  ensued.  Very  few  plutei  were  obtained. 
The  results  of  Godlewski  were  fully  confirmed  by  Baltzer.^ 
The  skeleton  was  as  in  Godlewski's  experiments  purely  maternal. 
He  confirms  the  result  of  Godlewski,  that  in  spite  of  the  par- 
ticipation of  the  paternal  chromosomes  in  the  development  no 
transmission  of  hereditary  characters  can  be  observed. 

1  Godlewski,  "  Untersuchungen  ueber  die  Bastardierung  der  Echinoiden- 
und  Crinoiden-familie,"  Archiv  f.  Eniwicklungsmechanik,  XX.  579,  1906;  Das 
V ererbungs problem  im  Lichte  der  Eniwicklungsmechanik  betrachtet,  Leipzig,  1909. 

2  Baltzer,  "Ueber  die  Beziehung  zwischen  dem  Chromatin  und  der  Entwick" 
lung  der  Vererbungsrichtuug  bei  Echinodermenbastarden,"  Arch.  f.  Zdlforschg., 
V,  497,  1910. 


294     Artificial  Parthenogenesis  and  Fertilization 


The  fertilization  of  the  eggs  of  the  sea-urchin  with  the' sperm 
of  molluscs  and  annelids  by  Kupelwieser  and  by  Godlewski 
(see  chap,  xxii)  did  not  lead  to  an}'  normal  larvae  and  cannot 
therefore  be  utilized  for  this  problem.  The  writer's  experiments 
on  the  cross  between  S.  franciscanus  ?  and  Chlorostoma  S 
(a  mollusc)  gave  maternal  plutei. 

2.  It  can  be  stated  as  a  general  fact  that  the  rate  of  cleavage 
in  hybrid  eggs  is  exactly  like  the  rate  found  in  the  development 
of  eggs  fertilized  with  sperm  of  their  own  species.     The  writer 
found  this  to  be  true  for  sea-urchin  eggs  fertilized  with  the 
sperm  of  starfish.     Moenkhaus  measured  the  rate  of  segmen- 
tation in  h^'brid  fish  eggs  and  found  that  the  rate  for  the  first 
cleavages  is  determined  by  the  egg.^     The  egg  of  Ctenolabrus 
segments  about  forty  minutes  after  impregnation  with  sperm 
of  its  own  kind,  while  the  egg  of  Batrachus  tau,  if  fertilized  with 
the  sperm  of  the  same  species,  segments  after  about  eight  hours. 
If  the  egg  of  Batrachus  be  fertilized  with  the  sperm  of  Ctenolabrus 
it  also  does  not  segment   until  after  eight  hours.^     I  have 
repeated  these  experiments  in  a  number  of  fish  hybrids  and 
confirmed  Moenkhaus'  results.     This  fact  proves  again  what  we 
stated  in  a  previous  chapter,  that  the  spermatozoon  does  not 
start  the  development  by  carrying  an  enzyme  or  catalyzer  into 
the  egg,  which  the  latter  needs  in  order  to  develop,  but  causes  the 
development  by  altering  the  surface  layer  of  the  egg.    If  the  seg- 
mentation of  the  egg  were  caused  by  an  enzyme  carried  into  the 
egg  by  a  spermatozoon,  the  rate  of  cleavage  of  slowly  develop- 
ing eggs  should  be  accelerated  by  a  spermatozoon  of  a  species 
developing  at  a  faster  rate.    The  egg,  however,  behaves  exactly  as 
we  should  expect  from  the  fact  that  the  spermatozoon  removes 
only  certain  obstacles  for  the  development  of  the  egg,  but  does 
not  cause  its  segmentation  by  carrying  an  activating  enzyme. 

•  Moenkhaus,  Am.  Jour.  Anat.,  II f.  29,  1904. 

-  The  acceleration  of  segmentation  which  Newman  observed  in  the  egg  of 
Fundulus  majali.'i  fertilized  by  the  sperm  of  Fuudulus  heterorlitus  is  too  small  to 
influence  our  conclusions. 


Artificial  Parthenogenesis  and  Heredity        295 

But,  while  the  rate  of  segmentation  is  thus  essentially  de- 
termined by  the  egg,  the  same  is  not  true  for  the  rate  of  the 
further  development  of  the  embryo.  This  can  be  shown  in 
comparing  the  time  of  appearance  of  certain  characters  in  the 
hybrids  and  pure  breeds  of  two  related  species.  Bancroft  and 
the  writer  measured  the  time  which  elapses  from  the  moment 
of  fertilization  to  the  time  of  appearance  of  the  heart  beat,  the 
circulation,  the  red  chromatophores  of  the  yolk,  the  black 
chromatophores,  etc.,  in  the  embryos  of  Fundulus  heteroclitus, 
Fundulus  majalis,  and  the  two  hybrid  forms  majalis  9Xhetero- 
clitus  S,  and  heteroclitus  2Xmajalis  S.  The  rate  of  development 
is  much  slower  in  the  pure  breed  of  majalis  than  in  the  pure 
breed  of  heteroclitus.  If  the  egg  of  majalis  is  fertilized  with 
the  sperm  of  heteroclitus  the  rate  of  segmentation  is  practically 
the  same  as  if  it  were  fertilized  with  the  sperm  of  majalis. 
The  appearance  of  certain  embryonic  characters  is,  however, 
accelerated  as  the  following  table  shows. 


TABLE  XXXIX 


. 

Beginning  of 

First  Appearance  of 

Breed 

Heart 
Beat 

Circula- 
tion 

Red  Yolk 
Chroma- 
tophores 

BlackYolk 
Chroma- 
tophores 

Black  Pig- 
ment of 
Ej'es 

Heteroclitus  $  X  Heteroclitus  S  . 
Heteroclitus  ?  X  Majalis  i  .... 
Majalis  $  X  Heteroclitus  6   .  .  .  . 
Majalis  ?  X  Majalis  $ 

4d.     2h. 
4d.  17  h. 

5d.  14  h. 
5d.  19  h. 

4d.  15  h. 
5d.  12  h. 
6d.  14  h. 
6d.  18  h. 

3d.   22  h. 
4d.   17h. 
6d.     8h. 
9d.   10  h. 

3d.   16  h. 
4d.     3h. 
od.   18  h. 
7d.   17  h. 

7d.     6h. 
8d.     2h. 
9(1.   20  h. 
9(1.    14  h. 

It  is  obvious  that  the  entrance  of  the  heteroclitus  sperm  into 
a  majalis  egg  causes  the  red  and  black  yolk  chromatophores 
to  appear  from  two  to  three  days  earlier  than  if  the  majalis 
sperm  enters  the  same  egg. 

This  may  be  accounted  for  by  the  fact  that  the  j^rocess  of 
development  of  the  embryo  is  essentiall}^  determined  by  the 
transformation  of  yolk  material  into  the  living  organs  of  the 


296     Artificial  Parthenogenesis  and  Fertilization 


embryo  and  of  the  yolk  sac,  and  that  the  velocity  of  this  re- 
action is  not  only  determined  by  the  mass  and  character  of 
the  yolk  in  the  egg,  but  also  by  the  mass  and  character  of  the 
sperm  nucleus  which  centers  the  egg.  Whether  or  not  this 
latter  material  consists  of  enzj^mes,  as  has  been  suggested, 
need  not  be  considered  in  this  place. 

From  this  it  is  also  clear  why  the  rate  of  development  in 
the  two  hybrids  Is  not  identical  although  the  constitution  of 
the  nuclei  is  identical  in  the  two  hybrids.  The  mass  and 
probably  the  quality  of  the  yolk  material  is  difTerent  in  the  two 
eggs  and  hence  the  velocity  of  development  of  the  embryos  are 
different. 

In  many  and  especially  heterogeneous  hybrids  still  another 
factor  is  to  be  considered,  namely,  that  the  foreign  spermato- 
zoon makes  the  egg  sickly  and  that  as  a  consequence  the  later 
development  of  the  embryo  is  often  retarded  in  comparison 
with  the  pure  breed. 

This  is  most  strikingly  the  case  in  the  cross  between  the 
sea-urchin  and  the  starfish.  As  I  pointed  out  long  ago  the 
larvae  die  mostly  in  the  gastrula  stage,  and  possibly  one  egg  in 
a  million  reaches  the  pluteus  stage.  The  development  of  the 
pluteus  is  in  such  cases  always  retarded. 

We  find  such  a  retardation  not  only  in  the  case  of  hetero- 
geneous hybridization,  but  occasionally  also  in  the  case  of 
crosses  between  closely  related  forms.  While  the  hybrid 
purpuratus  $  and  franciscanus  $  is  vigorous,  the  hybrid  fran- 
ciscanus  $  and  purpuratus  6  is  sickly  and  reaches  the  pluteus 
stage  only  rarely  and  slowly. 

3.  There  are  other  facts  which  indicate  that  in  heterogeneous 
hybrids  the  spermatozoon  has  merely  a  developmental  but  not 
a  hereditary  effect.  Moenkhaus  found  that  the  eggs  of  bony 
fishes  can  easily  be  impregnated  with  foreign  sperm,  but  that 
they  do  not  develop  very  far.  Thus  he  states  that  the  hybrids 
between  Menidia  and  Fundulus  heteroclitus  ''never  go  beyond 


Artificial  Parthenogenesis  and  Heredity        297 


the  closure  of  the  blastopore."  I  have  been  able  to  rai.e  the 
hybrid  between  Fundulus  heterocKlus  ?  and  Menidia  Clem,- 
labrus,  and  Stenotomus  i  in  large  numbers  beyond  this  stage 
These  hybrids  lived  a  month  or  longer,  formed  hearts,  blood 
vessels,  eyes,  and  fins,  but  never  hatched.  With  a  few  excep- 
tions no  circulation  was  ever  established  although  the  lieart 
beat  for  weeks. 


days^ofd.  s\'o;^Sti?el^.^Se"rn:i%^£?t^^^^^^  '  -^   -^-^•'^-  ^.  twenty 

Fig.  87  shows  a  three-weeks-old  hybrid  of  Fundulus  hefero- 
clitus  ?  and  Menidia  $.     The  pure  breed  of  Fimdtdus  hetero- 
clitus,  of  the  same  age,  were  already  hatching.     The  hybrid 
embryos  had  formed  the  pigment  characteristic  for  the  pure 
breed  of  Fundulus  heteroclitus.     But  the  anomalies  of  the  em- 
bryo are  very  obvious.     The  embryo  is  rather  small,  owing  to 
the  slowness  with  which  these  hybrids  digest  the  yolk.     Its 
eyes  are  abnormal  and  approach  the  cyclopean  condition.     In 
many   specimens   only  irregular  masses   of  pigment   indicate 
where  the  eyes  should  be.     The  head  is  comparatively  small 
and  not  bent  as  is  characteristic  for  the  pure  breed.     The  heart 
is  developed  but  corresponds  to  an  early  stage  in  the  develop- 
ment.    It  beats  regularly  and  at  an  almost  normal  rate.     The 


298     Artificial  Parthenogenesis  and  Fertilization 


main  blood  vessels  exist  and  haemoglobin  is  formed,  but  the 
creeping  of  the  pigment  cells  upon  the  blood  vessels  does  not 
take  place. 

Years  ago  I  found  that  the  marking  of  the  yolk  sac  of 
Fundulus  and  of  the  embryo  is  caused  by  the  creeping  of  the 
chromatophores  upon  the  blood  vessels.  I  showed  that  this 
phenomenon  is  due  to  a  tropism  which  depends  upon  the 
circulation.  When  the  circulation  was  suppressed  pigment 
was  formed  but  the  chromatophores  did  not  creep  upon  the 
blood  vessels.  At  that  time  I  had  succeeded  in  suppressing 
the  circulation  for  a  few  days.^  In  the  new  experiments  the 
hybrid  embryos  lived  for  a  month  or  more  with  pigment,  but 
without  a  circulation.  They  demonstrate  the  correctness  of 
my  former  statement,  inasmuch  as  the  creeping  of  the  chro- 
matophores upon  the  blood  vessels  did  not  take  place.  They 
also  confirm  the  statement  that  the  formation  of  pigment  cells 
is  independent  of  the  circulation.  Newman  seems  to  hold 
the  opposite  view,  but  he  evidently  did  not  test  his  assertion 
experimentally. 

These  hybrids  are  also  smaller  than  the  pure  breeds  of  the 
same  age,  owing  to  the  fact  that  the  yolk  is  less  rapidly  digested 
in  the  hybrids  than  in  the  pure  breeds.  This  is  a  very  impor- 
tant link  in  our  conclusions  on  heredity.  The  development  of 
hereditary  characters  is  the  result  of  the  nature  and  the  velocity 
of  chemical  reactions  between  the  mass  of  yolk  on  the  one  hand 
and  the  substances  in  the  nucleus,  especially  the  chromo- 
somes, on  the  other.  If  two  closely  related  forms  be  crossed, 
the  chemical  reactions  need  not  be  materially  different  in  quality 
and  velocity  from  those  of  the  pure  breeds.  But  when  distant 
forms  are  crossed  it  is  to  be  expected  that  greater  differences 
in  the  nature  and  the  rate  of  chemical  reactions  will  be  found 
and  the  outcome  will  be  pathological  embryos  and  very  likely 

1  Loeb,    Jour.  MoTphol.,  VIIl,  161,  1893;  Mechanistic  Conception  of  Life,  p.  105 
1912. 


Artificial  Parthenogenesis  and  Heredity 


299 


a  suppression  of  the  paternal  influence.     The  disturbance  is 
the  same  in  practically  all  the  heterogeneous  hybrids.     I  liave 
also  produced  the  crosses  between  Ctenolabrus  6  and  Fundulus 
heteroclitus    9    and    between    Stenotomus    6     and     Fmidulus 
heteroclitus    ?.      The   result    was    about    similar   to    the    one 
described  here.     In  all  cases  there  was  a  consumption  of  yolk, 
development  of  an  embryo,  of  pigment,  of  a  heart  beat,  of  eyes,' 
lenses,  ears,  fins;  but,  with  rare  exceptions,  there  was  no  circula- 
tion.    The  number  of  relatively  good  embryos  was  very  large 
in  the  cross  between  Fundulus  heteroclitus  $  and  Menidia  6 
(where  about  90  per  cent  formed  embryos  that  lived  for  about 
a  month) ;   it  was  much  smaller  in  the  cross  between  Fundulus 
heteroclitus  ?  and  Ctenolabrus  $.      One  word  should  be  said 
m  regard  to  the  development  of  the  head  in  these  embryos. 
In  later  stages  it  is  often  abnormally  small  in  comparison  with 
the  body.     The  reason  for  this  is  that,  although  at  first  the 
head  of  these  heterogeneous  hybrids  develops  normally,  sooner 
or  later  its  development  stops  and  often  phenomena  of  degenera- 
tion set  in,  especially  in  the  eyes.     The  body  of  such  larvae, 
however,  continues  to  grow. 

All  these  hj^brid  larvae  between  Fundulus  ?  and  Menidia  S 
were  in  reality  pure  breeds,  namely  Fundulus  heteroclitus 
larvae  whose  development  was  retarded  through  some  inter- 
ference with  the  normal  chemical  reactions  in  the  egg;  and  the 
abnormalities  described  were  in  no  way  hybrid  characters. 
This  is  proved  by  the  fact  that  the  writer  was  able  to  obtain 
similar  larvae  by  putting  the  eggs  of  the  Fundulus  fertilized 
with  Fundulus  sperm  under  abnormal  conditions. 

All  the  facts  known  about  heterogeneous  hybridization  point 
to  one  conclusion:  namely,  that  in  these  cases  fertilization  is 
really  a  case  of  artificial  parthenogenesis. 

4.  Bataillon  had  observed  that  if  the  eggs  of  toads  are 
fertilized  with  the  sperm  of  frogs  the  eggs  die  very  early  without 
giving  rise  to  tadpoles.     G.  Hertwig,  however,  found  that  if  the 


300    Artificial  Parthenogenesis  and  Fertilization 


spermatozoa  of  the  frog  are  first  treated  for  a  longer  period 
with  a  strong  radium  bromide  preparation,  so  that  they  are 
no  longer  able  to  fuse  with  the  nucleus  of  the  egg,  they  can 
cause  the  toad  egg  to  develop  into  tadpoles  which  are  able 
to  live  as  long  as  from  three  to  five  weeks  and  which  are 
almost  normal.  In  this  case  the  foreign  spermatozoon  had 
only  a  developmental  effect  and  the  resulting  larva  was  prac- 
tically produced  by  artificial  parthenogenesis.^ 

5.  Combination  of  artificial  parthenogenesis  and  hybridiza- 
tion by  sperm. — Herbst  has  made  the  very  interesting  experi- 
ment of  fertilizing  eggs  with  the  sperm  of  a  different  species 
after  the  eggs  had  been  treated  with  a  fatty  acid.  In  this 
case  he  obtained  eggs  in  which  the  maternal  characters 
predominated.^  He  crossed  Sphaerechinus  ?  with  Strongylo- 
centrotus  $.  The  skeleton  of  the  plutei  of  these  two  forms  is 
typically  different  and  it  is  therefore  easy  to  follow  the  paternal 
influence  in  the  larvae.  Herbst  proceeded  in  the  following 
manner.  The  eggs  of  Sphaerechinus  were  first  treated  with 
50  c.c.  sea-water+3  c.c.  N/10  acetic  acid.  A  large  percentage 
of  these  eggs  formed  no  typical  fertihzation  membrane.  As  far 
as  I  can  learn  from  his  description,  the  eggs  formed,  however,  a 
fine  gelatinous  film  around  the  periphery.  These  eggs  had  there- 
fore undergone  a  peripheral  change  which  started  their  develop- 
ment. If  after  one  and  one-half  hours  sperm  of  Strongylocentrotus 
lividus  was  added  these  eggs  could  be  fertilized.  The  plutei  which 
were  thus  produced  were,  however,  much  more  like  the  pure 
Sphaerechinus  plutei  than  those  developing  from  the  eggs  which 
had  been  fertilized  with  the  same  sperm  but  without  having 
undergone  the  parthenogenetic  treatment  with  the  fatty  acid. 
The  explanation  for  this  result  was  found  by  Herbst  from  an 
analysis  of  the  nuclei  of  these  hybrids.    At  the  time  he  added  the 

iG.  Hertwig,  "Parthenogenesis  bei  Wirbeltieren,  hervorgerufen  durch  art. 
fremden,  radiumbestrahlten  Samen,"  Arch.f.  mikr.  Anat.,  LXXXI,  Abt.  2,  1913 

2  Herbst,    "  Vererbungsstudien,"  IV,   Archiv  f.   Entwicklungsmechanik,    XXII 
475;   1906;   V,  ihid.,  XXIV,  185,  1907;   VI.  ihid.,  XXVII,  266,  1909. 


Artificial  Parthenogenesis  and  Heredity        301 


sperm  to  the  eggs  the  nuclei  of  the  latter  had  begun  to  undergo 
an  increase  in  size  in  consequence  of  the  previous  treatment 
with  fatty  acid.  In  a  large  number  of  such  eggs  Herbst  found 
that  the  chromatin  of  the  sperm  nucleus  had  undergone  a  mi- 
totic division  and  modification.  Herbst  considers  it  likely 
that  this  is  responsible  for  the  partial  elimination  of  the  heredi- 
tary influence  of  the  sperm. 

It  is  very  interesting  that  Herbst  found  plutei  which  on 
one  side  of  their  bodies  were  purely  maternal  while  on  the  other 
side  the  paternal  influence  was  noticeable.  This  he  explains 
on  the  assumption  that  the  two  first  cleavage  cells  received 
a  different  amount  of  paternal  chromatin,  or,  more  correctly, 
different  paternal  chromosomes.  In  this  way  the  results  of 
Herbst  are  in  harmony  with  the  modern  results  concerning 
the  role  of  the  chromosome  in  heredity. 

Tennent  has  produced  results  similar  to  those  of  Herbst  by 
a  different  method,  namely,  by  modifying  the  concentration  of 
HO  ions  in  the  sea-water.  These  experiments,  however,  have 
no  relation  to  the  problem  of  artificial  parthenogenesis  and 
therefore  need  not  be  discussed  in  this  connection. 


XXXII 

CAN  AN  EMBRYO  DEVELOP  FROM  A  SPERMATOZOON  ? 

Leeuwenhoek,  the  discoverer  of  the  spermatozoon,  had  the 
idea  that  it  was  the  future  embryo.     Accor(nng  to  this  the  egg 
was  only  the  nutritive  medium  on  wliich  the  spermatozoon 
would  develop  into  the  embryo.     The  observations  on  natural 
and  artificial  parthenogenesis  have  put  an  end  to  such  a  view. 
It  had  been  shown  by  Boveri  and  confirmed  by  others, 
especially  Delage,  that  if  an  unfertilized  egg  be  divided  into 
two  fragments,  the  one  with,  the  other  without,  a  nucleus, 
the  enucleated  fragment  can  also  develop  if  a  spermatozoon 
enters.     This  case  of  development  of  an  enucleated  fragment 
of  egg  protoplasm  with  a  spermatozoon  has  been  utilized  to 
revive  the  idea  that  the  egg  was  only  the  nutritive  medium  for 
the  development  of  the  spermatozoon.     Against  such  a  con- 
clusion may  be  urged  the  fact  established,   especially  by  the 
work  of  E.  B.  Wilson,  Conklin,  R.  Lillie,  and  others,  that  the 
protoplasm  of  the  egg  and  not  its  nucleus  is  the  embryo  and 
that  the  protoplasm  of  the  unfertilized  egg  may  be  considered 
a  rough  preformation  of  the  later  embryo.     The  entrance  of  a 
spermatozoon  into  an  enucleated  fragment  of  an  egg  would 
simply  cause  the  fragment  to  run  its  course  of  development 
instead  of  undergoing  the  early  disintegration  to  which  it  would 
otherwise  be  doomed. 

Nevertheless  the  question  remains  whether  it  might  not 
after  all  be  possible  to  raise  an  embryo  from  a  spermatozoon,  if 
the  latter  were  transferred  to  a  suitable  medium. 

J.  de  Meyer  investigated  the  question  whether  it  is  neces- 
sary that  the  spermatozoon  should  come  in  contact  with  the 
cytoplasm  of  the  egg  in  order  to  undergo  the  first  phases  of  its 
normal  evolution.^     He  used  the  sperm  of  Echinus  microtubcr- 

»  J.  de  Meyer,  Arch,  de  Biologie,  XXVI,  G5,  1911. 

303 


304     Artificial  Parthenogenesis  and  Fertilization 


culatus,  which  he  placed  in  sea-water  containing  an  extract  of 
the  eggs  of  the  same  species  and  found  that  under  these  condi- 
tions the  spermatozoa  swelled  so  as  to  lose  completely  their 
normal  appearance.  The  tail  remained  unchanged,  but  the 
cytoplasmic  covering  of  the  head,  the  middle  piece,  and  the 
chromatic  portion  of  the  head  all  seemed  to  swell;  and  in  some 
cases  an  indistinct  vesicular  structure  was  seen  which  stained 
a  little  stronger  than  its  surroundings,  and  seemed  to  be  a 
nucleus.  He  concludes  that  incomplete  as  his  results  may  be, 
they  give  a  right  to  conclude,  ''that  the  male  just  as  the  female 
cell  is  capable  of  evolution  under  the  influence  of  external 
agencies." 

Loeb  and  Bancroft  undertook  experiments  to  see  whether 
the  spermatozoon  could  be  caused  to  develop  in  vitro  on  suitable 
nutritive  media.^  Their  experiments  were  carried  on  on  the 
sperm  of  the  fowl.  The  sperm  was  removed  aseptically.  Only 
the  sperm  contained  in  the  lower  portion  of  the  vas  deferens 
was  used.  It  was  kept  in  a  sterilized  moist  chamber  at  about 
39°  C,  but  was  always  used  soon  after  its  removal  from  the 
animal,  not  later  than  three  hours  after  it  was  taken  out.  The 
media  used  for  the  culture  of  the  spermatozoon  were:  egg 
yolk,  egg  albumen,  chicken  blood  serum,  and  m/6  and  m/10 
Ringer  solutions.  Slides,  cover  glasses,  and  instruments  were 
sterilized  in  a  flame  and  small  hanging  drops  of  the  various  media 
were  inoculated  with  the  spermatozoa.  The  cover  glasses  were 
inverted  over  hollow  slides  and  sealed  with  a  vaseline  and 
paraffin  mixture.  In  a  few  cases  the  eggs  were  broken  into 
glass  vessels  and  small  quantities  of  sperm  injected  into  the 
yolk  with  a  capillary  pipette.  After  stated  intervals  yolk  and 
sperm  were  taken  out  for  examination  with  a  capillary  pipette. 

When  the  spermatozoa  of  the  fowl  are  observed  in  a  hanging 
drop  of  white  of  egg,  kept  at  about  40°  C,  the  first  change  is 
seen  after  fifty  or  sixty  minutes.     It  consists  in  the  collection 

Loeb  and  Bancroft,  Jour.  Exper.  Zool.,  Xll,  381,  1912. 


Embryogenesis  from  the  Spermatozoon  305 


of  a  small  amount  of  some  substance  having  a  low  refractive 
index  about  the  middle  pieces  of  some  of  the  spermatozoa.     In 
favorable  cases  as  many  as  60  per  cent  of  tlie  spermatozoa  ma>' 
undergo  this  change.     At  this  time  many  of  these  sperniatozoa 
are  still  swimming.     During  the  course  of  the  next  few  hours 
these  lowly  refractive  areas  increase  in  size  until  they  iire  about 
half  as  long  as  the  sperm  head  and  acquire  a  fairly  distinct 
ellipsoidal  outline.     Then  in  many  cases  the  sperm  head  can 
be  seen  to  be  bent  in  a  horseshoe  or  spiral  shape,  and  to  be 
included  in  the  wall  of  the  vesicle,  which  has  now  become 
spherical,    wliile   the   tail    of   the   spermatozoon   still    remains 
unchanged  or  has  disappeared  without  taking  any  part  in  the 
transformation.     The  next  change  is  an  increasing  indistinct- 
ness in  the  sperm  head,  and  an  increasing  refractive  power  of 
the  whole  vesicle  so  that  it  can  hardly  be  discriminated  at  all 
in  the  albumen.     It  is  not  possible  to  follow  the  process  farther 
in  unstained  material. 

In  some  cases  these  vesicles  instead  of  being  spherical  stretch 
out  along  the  whole  side  of  the  sperm  head,  or  may  become 
entirely  disconnected  from  the  spermatozoon. 

If  yolk  is  used  as  a  culture  medium  for  the  sperm  essentially- 
the  same  phenomena  occur;  and  in  the  various  Ringer  solutions 
vesicles  containing  the  sperm  heads  are  also  formed,  but  in  the 
Ringer  solution,  as  a  rule,  the  steps  in  the  formation  of  these 
vesicles  could  not  be  seen  without  staining. 

When  the  hanging  drops  are  fixed  in  Flemming's  fluid  and 
stained  and  examined  in  Herla's  vesuvin  and  malachite-green 
mixture,  it  can  be  seen  that  in  its  early  stages  the  vesicle  has 
distinct  walls  and  a  homogeneous  unstained  fluid  of  a  low 
refractive  index  in  its  interior.  This  fluid  is  possibly  water  and 
this  would  account  for  the  fact  that  the  vesicle  is  consj)icuous 
in  albumen  and  yolk,  but  invisible  in  Ringer  solution. 

The  vesicle  seems  to  be  formed  by  the  imbibition  of  water 
by  the  very  thin  protoplasmic  envelope  of  the  sperm  head  and 


306    Artificial  Parthenogenesis  and  Fertilization 

middle  piece.  For  after  the  formation  of  the  vesicle,  head, 
tail,  and  middle  piece  are,  so  far  as  can  be  seen,  unchanged. 

When  the  vesicle  has  reached  its  full  size,  the  material  of 
which  its  surface  is  composed  seems  to  wet  the  sperm  head 
very  easily.  For  in  the  next  stage  the  sperm  head  is  in  contact 
with  the  wall  of  the  vesicle  along  its  whole  length,  and  the 
vesicle  has  usually  assumed  a  more  or  less  spherical  shape. 

Up  to  this  point  the  transformations  were  found  to  take 
place  in  the  same  way  in  all  the  media  employed,  but  in  the 
various  Ringer  solutions  the  transformation  went  no  farther 
than  this,  even  when  the  spermatozoa  were  left  in  the  solutions 
for  forty-eight  hours  and  longer.  In  the  yolk  and  albumen, 
however,  the  development  toward  the  formation  of  a  nucleus 
went  a  little  farther. 

After  the  spermatozoa  have  been  left  in  contact  with  the 
culture  medium  for  about  eighteen  hours,  many  fairly  normal- 
looking  nuclei  appear,  in  which  the  chromatin  is  all  present  in 
the  shape  of  discrete  particles  resting  on  the  nuclear  wall,  and 
in  which  no  linin,  or  but  very  small  amounts  of  it,  can  be  seen. 
It  would  seem  probable  that  the  chromatin  in  these  nuclei 
is  derived  by  a  condensation  of  the  uniformly  distributed 
chromatin  of  the  previous  stage,  though  it  is  possible  that  in  a 
certain  number  of  cases  the  sperm  head  breaks  up  into  chromatin 
particles  without  a  previous  complete  solution. 

From  these  experiments  we  may  conclude  that  in  yolk  and 
white  of  egg  the  spermatozoon  undergoes  the  transformation 
into  a  nucleus.  We  have  not  noticed  any  mitosis  or  aster 
formation  and  we  are,  therefore,  not  yet  in  a  position  to  state 
that  the  spermatozoon  can  undergo  mitosis  outside  the  egg.  It 
is  therefore  at  present  impossible  to  state  that  the  spermatozoon 
is  capable  of  development  into  more  than  a  nucleus. 

The  question  whether  or  not  a  spermatozoon  can  give  rise 
to  an  embryo  without  an  egg  cannot  yet  be  answered  in  the 
affirmative. 


INDEX 


INDEX 


Acetate,  ethyl,  fertilizing  effect  of,  66. 

Acids,  chemical  constitution  and  rela- 
tive physiological  efficiency  of.  133 
ff.;  monobasic  fatty,  fertilizing  ef- 
fect of,  67  ff. 

Acmaea,  267. 

Activation,  osmotic,  of  the  unfertilized 

egg,  57  ff. 
Amphitrite,  12,  63,  265. 
Antedon,  293. 
A  pus,  46. 

Arbacia     18  ff.,    27  ff.,    50,    54.    56,    65, 
69,  71.  73,  88,  92  ff.,  122,  127,  147  ff 
159,    165.    186  ff..    194.    197.    201  ff" 
213,    219,    237,    252.    281;     osmotic 
activation    of,    57  ff . ;      temperature 
coefficient,   for  the  velocity  of  seg- 
mentation  in,    32  ff. ;     of   oxidations 
in,  33. 
Arbacia  punctulata.  222. 
Arbacia  pustulata,  221  ff. 
Arbacia  pustulosa,   26  ff. 
Arrhenius,  101. 
Artemia,  46. 

Ascaris  megalocephala,  216. 
Asterias,  12,  197,  203,  281.  284;    sperm 
of,    used    to    fertilize    eggs    of    sea- 
urchin,  225  ff.,  291  ff. 
Asterias  forbesii,   63,   243,   252  ff.,   255 
Asterias  glacialis,  254. 
Asterina,    7,    197,    203,    216,    264,    288' 
artificial  parthenogenesis  In  the  eggs 

Astrospheres,  formation  of,  74.   124  ff. 
Auduinia,  230. 

Baltzer.  293. 

Bancroft,  16,  273,  275,  295.  304 

Barthelemy,  45  ff..  47. 

Bases,  activation  of  the  unfertilized 
egg  by,  147  ff. 

Bataillon,  217,  235,  272  ff..  299. 

Batrachus  tau,  294. 

Berkeley.  Lord,   131. 

Berlepsch,  von.  44. 

Blastomeres,  fertilization  of.  with 
sperm,  237  ff. 

Blood  and  cell  extracts,  foreign,  ferti- 
lizing effect  of,  191  ff. 

Bohn,  G.,  188. 

Bohr,  38. 

Bombyx  mori,  44  ff.,  47  ff. 

Bonnet,  43. 

Boursier,  47. 

Boveri,  3  ff..  29,  51  ff.,  236.  240  ff..  303. 

Brachet.  273. 

Bresslau.  E.,  44. 

Bufo  americanus,  274. 

BuUot.  265. 

Cell  division,  prevention  of,  71,  78.  85- 

process  of.  19ff..  25  ff. 
Centrosomes.  formation  of,  74,  124  ff 


4,  6.  53  ff..  63.  231.   245. 


274. 
19  ff. 


299. 

in   the 
123  ff. 


partheno- 


Chaetopterus, 
263. 

Chlorostoma,  2.  230.   294 

Chiton.  203. 

Chorophilus  feriarum. 

Cleavage,  process  of, 

Cohen.  101. 

Conklin.  29  ff.,  303. 

Connstein,  39. 

Ctenolabrus,  294,  297 

Cumingia,  10,  269. 

Cytological  changes 
genetic  egg,  113  ff.,  ._, 

Cytolysis,  mechanism  of,  188  ff  •  mem- 
brane formation  and,  173  ff  •  in- 
duced, with  bile  salts,  177  ff  •  '  with 
distilled  water.  183  ff. ;  with  fat- 
solvents  181  ff  ;  with  higher  fatty 
acids,  18o;  with  increase  of  tempera- 
i^^«  185;  with  isotonic  solutions, 
18b  tf.;  with  saponin,  115,  174  ff  • 
with  soaps,  178  ff. 
"Cytolysis,   black,"   89  ff.;     white.   91. 

DeGeer,  43. 

Dehome,  273. 

Delage    16.  130,  170  ff.,  254,  303. 

Delages  solution,   170  ff 

De  Meyer,  J.,  303. 

Dendrostoma,   191  ff.,   196. 

^??;^!?P^^^^'  affected  by  temperature 
/3  ff. ;  comparison  of  rate  of,  in  pure 
and  hybrid  forms,  295  ff  •  is  it 
possible  without  membrane'  forma- 
o?^fT^^  ^'ithoiit  corrective  factor. 
219  ff.;  morphology  of,  17  ff.;  tem- 
perature coefficient  of,  101  ff  •  nre- 
:^'|^ion  of,  by  lack  of  oxygen,  25  ff.. 
77  ff. ;  by  addition  of  KCN  26  ff 
77  ff.  • 

Dewitz,  J.,  49. 

Disintegration,  after  membrane  forma- 

ri-^^T  P^®y^^^^®^  o'-    ^y  addition  of 
KCN  or  lack  of  oxygen,  26  ff     77  f 
85    ff. ;    by   exposure    to    hypertonic 
solution.    95   ff.;    of  fertilized    eggs 
after  treatment  with  aerated  hyper- 
tonic sea- water.   89   ff. ;    process   of 
after  artificial  membrane  formation 
at  room  temperature,   74  ff.  •   at  low 
temperature,  76. 

Driesch,  240. 

Dzierzon,  43  ff. 


303. 


Echinua,  170.   188. 

Echinus   mirrotuberculatus 

Elder.  217,  22S. 

Electrical  conductivity  of  fertiUzed  aiu 

unfertilized  eggs.    122  ff. 
Embryogenesis  from  tin 

303  ff. 
Erdmann.  Mi.ss.  29. 


spermatozoon. 


309 


310     Artificial  Parthenogenesis  and  Fertilization 


Fertilization,  heterogeneous.  2,  225  fT., 
291  ff.;  membrane  of,  see  Membrane 
formation;  oxidation  and.  25  ff. ; 
preservation  of  the  life  of  the  egg  by 
the  act  of,  281  ff. ;  witli  sperm  and 
artificial  parthenogenesis  in  the  same 
egg,  283  ff. 

Fertilizing  effect,  of  foreign  blood  and 
foreign  cell  extract.  191  ff.;  of  sperm 
extract,  201  ff. 

Fischer.  03.  205. 

Flemming's  fluid.  305. 

Francotte,  265. 

Fucaceae,  211 . 

Fucus  vesiculosus,  211  ff. 

Flihner,  134.  139. 

Fuiululus,  271. 

Fundulus  heteroclitus,  hybrid  forms  of, 
and  Ctenolabrus,  297,  299;  and  F. 
majalis,  295;  and  Menidia.  296  ff., 
299;    and   Stenotomus,  297,  299. 

Fundulus  jtiajalis,  hybrid  forms  of,  and 
F.  heteroclitus,  29.5. 

Garrey,  AV.  E.,  58  ff.,  129. 

Gelatinous  film,  formation  of,   19,  97, 

213,  223. 
Gephyrea,  9,  191. 
Germination    of    oil-containing    seeds, 

hydrolvtic  processes  in,  39  ff. 
Gies,  W.'J.,  202  ff. 

Godlewski.  2,  25,  143,  231,  254,  293  ff. 
Green,  C.  W.,  52. 
Guyer.  271  ff. 

Hagedoorn,  146. 

Harvey,  92,  121,  150,  217. 

Hasselbalch,  38. 

Henneguy,  273. 

Herbst,  71.  73,  174.  187  ff.,  223,  .300  ff. 

Hereditary  characters,  transmission  of, 
291  ff. 

Heredity  in  artificial  parthenogenesis, 
291  ff. 

Herla's  vesuvin  and  malachite-green 
mixture,  305. 

Herold,  45,  47. 

Hertwig,  Gunther,  229,  299  ff. 

Hertwig,  O.,  3,  229. 

Hertwig,  Paula,  229. 

Hertwig,  R.,  49  ff.,  .54. 

Heteronereis,  248. 

Hindle,  E.,  123,  125.  237. 

Holbom,  129. 

Hoyer,  39  ff. 

Hybridization,  combination  of  arti- 
ficial parthenogenesis  and,  300  ff. ; 
heterogeneous,  comparison  of  rate 
of  first  appearance  of  certain  char- 
acters in,  295  ff . ;  in  echinoderms, 
225  ff..  291  ff.;  in  teleosts,  295  ff.; 
in  vertebrates,  299  ff. 

Hypertonic  solution,  action  of,  after 
membrane  formation.  95  ff. ;  activat- 
ing effect  of.  57  ff.,  1.59  ff.;  irre- 
versibility of  corrective  effect  of. 
110  ff.,  119  ff.,  151;  nuclear  divisions 
in,  50  ff. 

Insects,  natural  parthenogenesis  in, 
43  fl. 

Isosmotic  solutions,  physiological  effi- 
ciency of,  127  ff. 


Kirbv,  43. 

Klebs,  278. 

Knaffl,  von,  185.  188  ff. 

Koppe.  173.  188. 

Kohlrausch,  129. 

Kostanecki.  267.  209. 

Kupelwieser.  2,  203  ff.,  230,  294. 

Kuschakewitsch.  275. 

Leeuwenhoek,  303. 

Lefevre.  7.  245.  263,  265. 

Leuckart,  44,  46. 

Lillie,  F.,  216,  2.30,  235  ff.,  263. 

Lillie,   R.,  9,    14,    121,    185  fl.,    252  ff., 

.303. 
Llovd,  Miss,  170. 
Loeb,  Leo.  1.  205. 

Lottia  gigantea,  157,  160,  210  ff.,  267  ff. 
Luimadia,  46. 
Lyon,  E.  P..  221  fl. 

Mactra,  230.  267.  269. 

Mathews,  A.P.,  12,  246.  255.  286. 

Maturation  divisions,  prevention  of, 
by  lack  of  oxygen  or  presence  of  KGN, 
27;    conditions  for,  243  ff. 

Maxwell,  S.  S.,  102. 

McClendon.  22,  121  ff.,  273. 

Mead,  4,  51  ff..  245,  263. 

Membrane  formation,  artificial,  effect 
of,  73  ff. ;  indticed,  by  foreign  blood 
and  foreign  cell  extracts.  191  ff.; 
by  haemolytic  agents,  68,  173  ff.; 
by  monobasic  fatty  acids,  67  ff . ; 
is  development  possible  without. 
219  ff.;  mechanism  of.  17  ff.,  207  ff.; 
temperature  coefficient  of,  146. 

Membrane  formation  and  cytolysis, 
173  fl. ;  by  means  of  bile  salts,  177  fl. ; 
of  distilled  water,  183  ff . ;  of  fat- 
solvents,  181  ff.;  of  higher  fatty 
acids,  185;  of  increase  of  tempera- 
ture, 185;  of  isotonic  solutions,  186 
ff. ;  of  saponin,  174  fl.;  of  soaps, 
178  fl. 

Menidia,  296  fl.,  299. 

Meyer,  Hans,  139. 

Meyerhof,  38,  71,  118. 

Moenkhaus.  294,  296. 

Moore,  A.  R.,  96,  218. 

Morgan,  4,  51.  53.  236,  241. 

Morphology  of  development,  some 
remarks  on  the.  17  ff. 

Morse.  131. 

Mytilus,  2,  203.  2.30. 

Neilson,  63,  253,  265. 

Nematus,  46. 

Nereis,  216,  230,  236,  247,  265. 

Neubauer,  134,  139. 

Newman,  298. 

Norman,  W.  W.,  51. 

Nuclear    division,    prevention    of,    by 

lack  of  oxygen  or  presence  of  KGN, 

26  ff..  85;    process  of,  19  ff. 
Nuclear  spindle  formation.  19  ff.,  192. 
Nuclei,  fusion  of,  in  hybridization,  293. 

Ophelia,  265. 
Overton,  36,  136,  139. 
Overton,  J.  B.,  277. 
Oxidations,  coefficient  of  rate  of,  161, 
106  ff.;   effect  of  NaOH  and  NH4OH 


Index 


311 


upon.  36  ff.;  rate  of,  2S  fr. :  suppres- 
sion of,  by  addition  of  KCX,  20,  71; 
temperature  coefficient  of,  33. 

Oxygen,  prolongation  of  the  life  of  the 
egg  by  lack  of,  or  addition  of  KCN, 
SoflF. 

Oxygen  consumption,  coefficient  of, 
162;  determination  of.  2(1  ff.,  114  ff.. 
152  ff.,  155  ff.,  161,  160  tr. 

Palla,  278. 

Parthenogenesis,  artificial,  and  fertili- 
zation with  sperm  in  the  same  egg. 
233  ff.;  and  heredity,  291  ff.;  and 
hybridization  by  sperm,  300  ff.; 
effect  of  the  agencies  of,  upon  oxida- 
tions, 113  ff.;  history  of  the  earlier 
experiments  on,  47  ff . ;  in  the  eggs 
of  annelids,  257  ff . ;  in  frogs,  271  ff . ; 
in  molluscs,  267  ff. ;  in  plants,  277  ff. ; 
in  the  sea-urchin,  65  ff . ;  in  the  star- 
fish, 249  ff. ;  induced,  by  bases,  147 
ff. ;  by  ethyl  acetate,  66;  by  foreign 
blood  and  cell  extracts,  191  ff. ;  by 
haemolytic  agents,  68  ff. ;  by  hyper- 
tonic solutions,  57  ff.,  159  ff.;  by 
monobasic  fatty  acids,  67  ff. 

Parthenogenesis,  natural,  in  insects, 
43  ff . ;   in  starfish,  255  ff . 

Parthenogenesis,  osmotic,  57  ff . ;  analy- 
sis of  method  of,  159  ff. 

Parthenogenetic  egg,  cytological 
changes  in,   113  ff..   123ff. 

Patella,  230. 

Permeabihty  of  fertilized  and  miferti- 
lized  eggs,  121  ff.,  136  ff.,  149  ff. 

Pfltiger,  275. 

Physiological  efficiency  of  isosmotic 
.solutions,  127  ff. 

Pieri,  201. 

Polistes,  46. 

Polynoe,  7,  8,  73,  149,  153,  244  ff.,  247, 
268,  281;    fertilization  of,  257  ff. 

Polyorchis,  130. 

Prostheceracus,  265. 

Protoplasm,  cleavage  of,  20. 

Psyche  helix,  43. 

Psychidae,  46. 

Pyrnopodia,  197. 

Quincke's  albumin  soap,  21. 

Radium,  effects  of.  on  sperm,  229. 

Rana,  272. 

Rana  fusca,   49. 

Rana  pipiens,  274. 

Rana  silvatica,  274. 

Rana  sphenocephala,   274. 

Ratzeburg,  43. 

Reaction  velocity,  effect  of  tempera- 
ture on,  26,  32  ff. 

Reaumiu",  43. 

Ries,  217. 

Ringer,  54,  62. 

Ringer  solution,  52.  110,  116,  147  ff., 
161.  263,  269.  304  ff. 

Robertson,  T.  B.,  21  ff.,  194,  200,  206, 
214  ff. 

Roux,  \V.,  49. 

Samassa,  25. 
Schmid.  45  ff. 
Scott,  265. 


Segmentation  of  the  egg.  20;  temi)(Ta- 
ture  coefficient  of  the  velocitv  of, 
32  ff. 

Sex,  determination  of,  in  the  frog,  275. 

Shearer,  71,  170. 

Siebold,  von,  43  If.,  47  ff. 

Sipunculus,  247. 

Snyder,  C.  D..  102. 

Solenobin,   43.   4(). 

Sperm  extract,  fertilizing  effect  of. 
201  ff. 

Spermatozoon,  action  of.  upon  the 
egg.  225  ff.;  effects  of  radium  upon, 
229. 

Sphaerechinus,  201  ff.,  223;  fertilized 
with  sperm  of  Stroiu/ylorentrotus,  300. 

Starfish  eggs,  artificial  parthenogenesis 
in,  249  ff.;  maturation  of,  243  ff.; 
natural    parthenogenesis    in,    255  ff. 

Stenotomus.  297,  299. 

Streaming  phenomena,  21. 

Stronyylocentrotus  franri.'icanua,  .50,  58 
ff.,  186,  198,  203,  206,  222,  228,  230; 
fertilized  with  sperm  of  molluscs, 
230,  294;  sperm  of,  used  to  fertilize 
eggs  of  sea-urchin,  292  ff.,  296. 

Strongylocentrotus  lividus,  114,  116,  221 
ff.;  sperm  of ,  ased  to  fertilize  Sphae- 
rechinus eggs,  300. 

Strongylocentrotus  purpuratus,  6,  8, 
56,  58  ff.,  61,  65  ff.,  71,  74,  77,  80. 
86  ff.,  89,  91,  93,  96,  99,  102  ff.,  107 
ff.,  110  ff.,  123,  127,  130,  132,  137, 
146,  150  ff.,  159,  178,  186,  190.  194, 
ff.,197ff.,200,  203ff..207,  209,213ff.. 
217,  219  ff.,  237,  240,  247.  252.  261. 
268,  287  ff . ;  blastula  stage  of,  23 ; 
concentration  of  HO  ions  necessary 
for  the  development  of,  34  ff . ; 
cytological  changes  in  the  eggs  of. 
123  ff.;  effect  of  lack  of  oxygen  or 
presence  of  KCN  on  development 
of,  30  ff.;  gastrula  stage  of,  23; 
hybridization  between,  and  Asterias, 
225  ff.,  291  ff.;  and  S.  franciscanus, 
292  ff.,  296;  increase  of  acid  content 
of  the  egg  of,  40  ff. ;  mechanism  of 
membrane  formation  in,  17ff. :  oxygen 
consumption  of  eggs  of,  27  ff..  1 14  ff.. 
152  ff..  155  ff.,  161  ff.,  166  ff . ;  pluteus 
stage  of,  23. 

Tangl,  38. 

Temperature,  cytolysis  produced  by 
increase  of,  185;  effect  of,  upoli 
development,   73  ff.,   252  ff. 

Temperature  coefficient,  determination 
of,  for  the  velocitv  of  segmentation. 
32  ff.;  of  development,  101  ff.;  of 
membrane  formation  by  means  of 
acids,  146;    of  oxidations.  33. 

Tennent,  293,  .301. 

Thalassema,  7,  73. 

Thalassema   inellitn.  24.5.  '2iV.\  ff. 

Thomson-Gibbs  principle,  2 IS. 

Tichomiroff,  A..  47  ff. 

Traube,  Moritz,  42. 

Van't  Hoff.  34.  101. 

Van't  Hoff  .solution.  34  ff..  00,   103  ff.. 

261,  267. 
Vespa,  46. 


312     Artificial  Parthenogenesis  and  Fertilization 


Warburg,  13,  26  ff..  28,  36,  71,  92,  114, 

116.   120.   121.   150,   169. 
Wartenberg,  39. 
Wasteneys,  10,  13  ff..  27  ff.,  31,  36  ff., 

85,    93,    114  ff.,    152,    169,    194,    197, 

249,  263.  269,  287. 
Weismann,  1. 


Wilson.  E.  B.,  303. 
AVinkler.  201  ff. 
Winkler's  method.  27,  152. 
Witte's  peptone,  200. 


Zuntz,  49. 


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